Final Report Summary - SITINPLANT (Sustainable innovation technology in plant nursery process improving plant quality and safety)
Executive summary:
The SITINPLANT project, funded by the European Commission under the 7th Framework Programme, started officially on 1 September 2008.
The aim of the SITINPLANT project was:
- to use mycorrhizae, antagonist bio-control microorganisms, during the acclimation phase of micropropagated rootstocks and in the nursery,
- to optimise an innovative micrografting technique.
During the two years of activity, the work achieved the following main objectives:
- Optimisation of the inoculation method on micropropagated rootstocks;
- Development of the correct interaction between mycorrhizae and antagonist biocontrol micro-organisms on controlling disease incidence and improving plant nutrient uptake;
- Optimisation of micrografting techniques;
- Optimisation of nursery growing techniques for micrografted and mycorrizated plants;
- Implementation and testing of the innovated plant materials under a range of pedo-climatic conditions.
Project Context and Objectives:
European and worldwide policies are increasingly working to limit the use of chemical substances for fertilisation and plant protection so as to reduce their negative environmental impacts and to increase soil fertility. In the plant nursery sector there is widespread use of chemical pesticides to repress plant pathogens and of synthetic fertilisers to stimulate plant growth. Both practices lead to a reduction in the complexity of the soil microflora and to a decline in overall plant quality. As a result, innovative nursery plant production processes are urgently needed to produce plants that are more tolerant of soil-borne pathogens and also more efficient in using the natural soil resources (minerals, water, etc.) in all phases of cultivation (in the tissue-culture laboratory for rootstock production, the nursery and the orchard).
During the two years of activity the work completed the following tasks:
T.2.1. Studies on physiological and molecular relationships between symbiont micro-organisms and plant root systems during the acclimatisation phase and in the nursery;
T.2.2. Effect of agronomic and environmental parameters on the establishment and continuance of symbiosis in plants grown in pots;
T.2.3. Plant-soil-microbe interactions in the rhizopshere influencing the rate of inoculation and efficiency of biocontrol;
T.3.1. Optimisation of techniques to inoculate micropropagated rootstocks at different growth stages;
T.3.2. Analysis of symbiotic effects on plant growth and quality in the nursery;
T.4.1. Definition of the optimum period for micrografting techniques in relation to biotisation;
T.4.2. Effects of biotisation of the rootstocks on micrografting success;
T.4.3. Effects of biotisation on grafting success of the plants grafted in the nursery;
T.5.1. Persistence of microorganisms in micrografted plants grown in pots;
T.5.2. Effect of different managements in the nursery on persistence of microorganisms in grafted plants on inoculated and non inoculated micropropagated rootstocks;
T.5.3. Evaluation of the growth and quality of micrografted and grafted plants on inoculated and non inoculated micropropagated rootstocks;
T.6.1. Production scheme of micropropagated and biotisated rootstocks;
T.6.2. Production scheme of micrografted and grafted fruit tree plants using inoculated micropropagated rootstocks grown in pots and in nursery;
T.7.1. Plan for use and dissemination of the foreground;
T.7.2. Project website.
Project Results:
WP2. Interaction of mycorrhizae and antagonist biocontrol micro-organisms.
The aim of this WP2 was to study the relationships between symbiont microorganisms and rootstock systems and to define the optimal agronomic and environmental conditions that foster the establishment and the continuance of symbiosis.
T.2.1 Study on the physiological and molecular relationships between symbiont microorganisms and the plant root system during the acclimatisation phase and in the nursery.
Part of the work in the T.2.1 was completed during acclimatisation. In particular, the results achieved in the first year showed that inoculation of the substrate with Trichoderma and Glomus of micropropagated rootstocks significantly decreases the percentage of dead plants during the acclimatisation phase. Inoculated plants without fungicide treatments reduced plant mortality. Because these results represent very important achievements, SME has already changed the plant production process reducing or eliminating the use of fungicides during the acclimatisation phase.
The activity carried out focused on: studies of gene expression and protein characterisation during the interaction between Trichoderma and plant roots in vitro. Trichoderma harzianum strain T-22 controls many plant pathogens through mycoparasitic interactions with the target fungal organism and it is believed that extracellular cell wall degrading enzymes produced by the antagonist play an important role in these interactions. It has also been reported that T. harzianum has the ability to directly enhance root growth and plant development in the absence of pathogens and it has been suggested that this is due to the production of a growth-regulating factor by the fungus. In spite of their theoretical and practical importance, the mechanisms responsible for the growth response due to the direct action of T. harzianum on plants have not been investigated extensively. All the above-cited observations indicate the versatility through which T. harzianum can manifest biological control activity. The application of T22 to in vitro cultured GiSeLa6® rootstocks (Prunus cerasus x P. canescens) during the rooting phase resulted in greater shoot lengths, numbers of leaves, numbers of roots and stem diameters. For this reason, we continued the experimentation on this rootstock, trying to explain the biochemical basis of plant-growth-promoting activity of T22. On the basis of the results of the previous experiments and of other research (RIF), we believe that the higher shoot and root growth in the plants inoculated with T22 could be due to a different phyto-hormonal balance or to medium acidification and redox fungal activity.
Hormonal regulation may be achieved by:
(1) a balance or ratio between hormones,
(2) opposing effects between hormones,
(3) alterations of the effective concentration of one hormone by another and
(4) sequential actions of different hormones.
For this reason not only is the hormone level very important per se but also their ratios. Considering the positive effects on shoot and root growth in plants inoculated with T22, we supposed that changes in auxin/cytokinins levels could be involved. It is known that a higher auxin to cytokinin ratio promotes root formation, whereas a higher cytokinin to auxin ratio promotes shoot-bud formation. Several lines of evidence indicate that while auxins stimulate root formation, cytokinins inhibit it.
Firstly, the results obtained demonstrate that the extraction methods, particularly complicated for phyto-hormones, were successful. In particular, two points during the extraction procedures are considered critical: maintenance of temperatures lower than 4°C during all the extraction steps and the concentration of the solvent mixture (not completely dry) with nitrogen flow to avoid the oxidation of hormones. For this latter point, a particular method using a nitrogen evaporator was designed and this allowed desiccation of all samples together and relatively fast. The competitive ELISA assay allowed a quantitative profile of the three hormones examined. In particular, the method discriminated well between IAA and TR-ZEA, whereas DH-ZEA was not present in either leaves or roots. This is normal as DH-ZEA is a precursor of TR-ZEA in the biosynthetic pattern used as 'zeatin storage' by plant cells. Thus, its absence suggests that the precursor TR-ZEA was not present before T22 inoculation and that all the TR-ZEA detected was produced ex novo after T22 exposure. The levels of IAA increased significantly after inoculation with T22 in both leaves and roots. This could explain the greater root and shoot growth in inoculated plants. Generally, IAA was higher in leaves but this is normal as it is produced mainly in leaves and then transported to the roots. As IAA regulates lateral foliar bud formation, it is directly related to the number of leaves. We have already demonstrated leaf number to be higher in T22-inoculated plants.
T.2.2 Effect of agronomic and environmental parameters on the establishment and continuance of symbiosis in plants grown in pots.
The experiments were carried out to evaluate during the growth cycle under pot conditions: a) the presence and persistence of microorganisms; b) the effects on persistence of agronomic parameters (inoculation method, peat type, pot shape, fungicide application); c) the effects of symbiosis on plant root conductivity; d) the effects of symbiosis and agronomic parameters on plant growth and mineral partitioning.
Two sets of plants produced in Greece were used for the Italian experiments:
- the first set was used to analyse microorganism persistence, effects of different substrates and pot shapes, and root conductivity. In particular, the rootstocks before the first acclimatisation phase (October 2008) were inoculated by mixing the substrate with microorganisms. The plants were then re-inoculated by water solution just before transfer to the nursery. All material was kept under greenhouse conditions of the SME Fitotechniki until March 2009 when it was transferred to Italy for further studies.
- the second set of plants was used to study the effects of inoculation methods and fungicide application. Those plants were acclimatisated in February 2009 (second acclimatisation period). Before the acclimatisation phase plants were inoculated with microorganisms by mixing into the substrate or by dipping in a water suspension. All plants were kept under regular greenhouse conditions of the SME Fitotechniki, including fungicide application until the transfer to Italy.
Concerning the first point (a), in Italy in pots the presence and persistence of biocontrol agents were checked on 10 plants per treatment and on three different sampling periods (April-July-December 2009). Qualitative and quantitative measurements were carried out on roots: infection and arbuscular percentage and intensity of colonisation for Glomus (590 samples-Biologic units); and on substrates: Colony Forming Units (CFU) for Trichoderma (680 samples-biologic units). Results showed that Glomus intraradices colonisation intensity for all rootstocks reached the highest values in July (2009) from 50 to 80%, the intensity percentages decreased successively in December (2009) as in the first measurement (April 2009). Glomus + Trichoderma (in Myrobolan 29C, GiSeLa®6 and OHF 19-89) or Glomus + Radiobacter (in GF677) treatments showed the highest colonisation intensities. There were significant differences in arbuscular abundance between control and treated roots, the values ranged between 0-10% and 10-60%, respectively. Trichoderma CFUs were higher in Myrobolan 29C and GiSeLa®6 rootstocks at the second sampling period (July) with values of 3-5 x 104 CFU x g-1. Glomus values reduced to 1 x 104 CFU x g-1in December 2009. In GF and OHF19-89, CFUs were fewer than previous rootstocks but remained constant or increased until December. Therefore, the general colonisation trend of Glomus intraradices and Trichoderma harzianum showed a peak in July followed by a decrease in December.
(b) Effect of inoculation methods: inoculum mixed into the peat substrate or dipping the plants in the water solution were evaluated for CFU of Trichoderma in GF677 rootstocks. In December, CFUs calculated for the dipping method increased to 2.5 x 104 CFU x g-1. This value was significantly higher than values obtained in the mixing method (1 x 104 CFU x g-1). Evaluation the effects of the two substrates types, enriched (TN) and impoverished (TP), were compared for mycorrhization rate and the persistence of Glomus and also on CFU capacity of Trichoderma in all rootstocks. Differences induced by the two substrates were not significant. The effects of two pot shape, round (VT) and square (VQ), were compared on mycorrhization rate and persistence of Glomus intraradices, in all rootstocks. Round pots enhanced root mycorrhization: intensity percentages were about 50% higher and arbuscular abundance x4 fold higher than in square pots, in July. Values decreased for round pots and differences became non significant in December. Colonisation for square pots remained lower, instead increased by about 10% from July to December. Glomus intraradices showed better colonisation in non fungicide treated plants, during the period April - July. Effect of the fungicide applications were evaluated on mycorrhization and persistence parameters of Glomus intraradices and on CFU capacity of Trichoderma for GF677 rootstocks during the period April-December 2009. Plants treated with fungicides showed, as for Glomus, CFUs of Trichoderma constant and significant higher of 30% in December. Microorganisms in plants without fungicide application developed better during the first phases of their colonisation. In addition in plants treated with Glomus intraradices and Trichoderma harzianum effects of symbiosis on plant root conductivity (Lp) showed an increase in Lp of about 30% above the controls. Further studies on the effect of symbiosis on plant growth parameters were carried out on 620 plants in the first and second samplings and on 80 plants in December. Results showed that: Glomus+Tricoderma (G+T) and Glomus+Radiobacter for GF677 (G+R) treatments enhanced plant growth by about 30 and 40% above the controls respectively. Positive results were also obtained using enriched substrates, in this case plant growth doubled.
T.2.3 Plant-soil-microbe interaction in the rhizosphere influencing the rate of inoculation and the efficiency of biocontrol.
The general objective of task 2.3 was to study the factors involved in the plant-soil-microbe relationship that increase the efficiency of biocontrol, in particular to evaluate the capability of biological agents against the most important fungal pathogens and the strategies to enhance these biocontrol activities.
UBAS and AUTH main activities were focused on: a) studies on the identification and characterisation of secondary metabolites involved in the bioactivity of Trichoderma; b) Trichoderma biocontrol efficiency against fungal pathogens (Fusarium, Phytium and Rhizoctonia).
Some Trichoderma spp. are considered to be important biocontrol agents against plant pathogen fungi since they use various defense mechanisms such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. Trichoderma harzianum is often used in liquid or dry formulations of bio-pesticides, bio-fungicides and bio-stimulants. These species also have the ability to produce volatile antifungal compounds that have a partial role in the inhibition of growth of plant pathogen fungi. In this part of the study we investigated the growth inhibition of three target fungi (Fusarium oxysporum, Pythium ultimum and Rhizoctonia solani) due to the production of volatile compounds by Trichoderma harzianum T22 and Trichoderma asperellum B1.
Potato dextrose agar (PDA) (20 mL) was poured onto Petri dishes. A 5 mm diameter agar disc was excised from the leading edge of a two-day-old pure culture of T. harzianum T22 and T. asperellum B1 cultures was placed at the centre of each agar plate. Next, a disc of the same size was taken from each target culture and likewise placed on another agar plate. The lids were removed, and target culture plates were immediately placed over each of the Trichoderma plates and held in place with adhesive tape. The head space prevented any physical contact between the pathogens and antagonistic fungi, so that the volatile compounds were formed and confined to the interior atmosphere of the two plates. For the control plate, only the targets were cultured on each Petri dish. The plates were randomised and incubated at 28±2ºC for 4 days. The diameters of target colony cultures were measured three times daily. Three replicate plates were set up for each treatment, and the experiment was repeated twice. The implication of the results was expressed as PIRG that considers the percentage inhibition of radial growth expressed as: %PIRG=((R1-R2))/( R1)*100, where: PIRG = percentage inhibition of radial growth; R1 = average value of the radial growth of the target in the absence of the antagonist (control); R2 = average value of the radial growth of the target in the presence of the antagonist. The experiments were repeated using other media (malt extract agar, minimal medium agar and Czapek-agar) but the best results were observed in PDA medium.
To investigate the bioactivity of Trichoderma against target microorganisms different tests were performed:
i) volatile test results of T. harzianum T22 and T. asperellum B1 against Fusarium oxysporum; ii) volatile test results of T. harzianum T22 and T. asperellum B1 against Rhizoctonia solani; iii) volatile test results of T. harzianum T22 and T. asperellum B1 against Rhizoctonia solani.
The presence of Trichoderma harzianum T22 and Trichoderma asperellum B1 induced an inhibiting effect on the target Fusarium oxysporum that probably was due to the production of volatile compounds since the hyphal contact was avoided in this test. Percentage inhibition radial growth (PIRG) of F. oxysporum in the presence of Trichoderma harzianum T22 was 49.4% while in the presence of Trichoderma asperellum B1 was 32.8%. The presence of Trichoderma harzianum T22 and Trichoderma asperellum B1, instead, induced a weak inhibiting effect on the target Rhizoctonia solani that probably was always due to the production of volatile compounds. Percentage inhibition radial growth (PIRG) of R. solani in the presence of Trichoderma harzianum T22 was 30.7% while in the presence of Trichoderma asperellum B1 was 12.5%. The presence of Trichoderma harzianum T22 induced an inhibiting effect on the target Pythium ultimum that we quantified with the PIRG value 30.9%. In contrast, Trichoderma asperellum B1 induced a very low inhibiting effect on Pythium and in this case the PIRG value was 4.7%. GC-MS in combination with solid-phase microextraction (SPME) will be used to explore the volatile secondary metabolites released by two fungal species grown on PDA. Extraction of the volatile substances will be performed by using a SPME system into the head space of the Erlenmeyer flask. Inside a cannula, the fibre will be passed through a septum and exposed to the volatiles derived from the cultures. After adsorption for 24-48 h, the fibre will be retracted into the needle and removed from the flask to perform gas-chromatographic analysis.
In addition, other studies were carried out on:
biological control of the Rhizoctonia solani and Pythium ultimum disease complex on GF rootstocks, using mixtures of Trichoderma harzianum T-22 with Trichoderma asperellum B1, Trichoderma viride and Pseudomonas fluorescens F113, during the acclimatisation period.
Disease severity: Trichoderma harzianum T22 combined with Trichoderma asperellum B1 significantly reduced the Rhizoctonia solani and Pythium ultimum mediated disease severity. In contrast, with the Trichoderma harzianum T-22 mixtures with Trichoderma viride and Pseudomonas fluorescens F113, there were no significant differences when compared with the positive control plants. The treatments seemed to be ineffective against the specific disease complex.
Plant growth promotion: compared to the control plants, Trichoderma harzianum T22 combined with Trichoderma asperellum B1 seemed to promote the plant growth characteristics measured (shoot weight, root weight and number of leaves). Among the treatments of the specific experiment, Trichoderma harzianum T22 combined with Pseudomonas fluorescens F113 showed increased levels of all plant growth characteristics only in the absence of the plant pathogens.
Colonisation experiments: Trichoderma harzianum T22 combined with Trichoderma asperellum B1 seems to be an excellent coloniser of the GF root system. There was no sign of competition between those two biocontrol agents, moreover the outcome from the interaction between them appeared as enhancing colonisation. Root samples taken two days after transplantation were 80% colonised by the specific biocontrol agents. The pronounced treatment revealed increased colonisation rates during the first ten days after transplantation - the crucial period for plant protection and disease severity - compared to Trichoderma harzianum T22 combined with Pseudomonas fluorescens F113 (moderate colonisation rates) and Trichoderma harzianum T22 combined with Trichoderma viride (low colonisation rates). In the turf colonisation experiments, the biocontrol treatments showed similar results. Specifically, the mixture of Trichoderma harzianum T22 and Trichoderma asperellum B1 was present in all three dilutions of the turf samples and was statistically differentiated compared to Trichoderma harzianum T22 combined with Pseudomonas fluorescens F113 (moderate turf colonisation activity) and Trichoderma harzianum T22 combined with Trichoderma viride (low turf colonisation activity).
WP3 - Optimisation of biotisation techniques on micropropagated rootstocks.
The aim of this WP3 was to define and optimise the most appropriate inoculation time and method, and to verify the effects of mycorrhysation on micropropagated rootstocks.
T.3.1 Optimisation of techniques to inoculate micropropagated rootstocks in the different growth stages.
We estimated: i) the effects of five different peat types: Kekkila, TS1, Klasman Plug Mix (Mix), Klasman Gold (Gold), and Havita; ii) the effects of treating or not with Trichoderma harzianum T22 and Glomus intraradices and Agrobacterium radiobacter K84.
The different method of inoculum applications were: a) mixing T. harzianum or G. intraradices with peat and incubating for a week before planting; b)mixing T. harzianum into peat and incubating for 20 days before planting; c) dissolving the inoculum into a solution and dipping the plants just before planting (dipping method). This was applied for Trichoderma and Glomus and A.radiobacter.
The rootstocks used were GF677, OHF19-89, Myrobolan 29C and GiSeLa® 6.
Concerning the different methods of inoculation applied it was concluded that the dipping method is better than the mixing one. This result has a positive impact on optimisation of rootstock biotisation techniques and also on reduction of the production costs for the SME. The dipping method is simpler, more economical and more efficient and increases the inoculum presence. Four weeks after inoculation, the G. intraradices root colonisation was not observed. Results showed that inoculation occurs later. In Italy, Glomus was detected after 6 months from inoculation. In general, regarding the effects of different peats on rootstocks inoculated with Glomus, Kekkila showed the best results for numbers of leaves, heights, shoot dry weights, except for GF677 rootstocks in which Havita peat performed better. Instead, Gold peat gave the best results for all rootstocks on root dry weight and TS1 on root and shoot ratio.
T.3.2 Analysis of symbiotic effects on plant growth and quality in the nursery.
The main objective was to study the effect of the mycorrhization on growth and plant quality as well as on the efficiency of nutrient uptake during rootstock development in the nursery.
During the reporting period, all those effects were evaluated in two different management systems. Conventional nursery management (Nursery MNGT) were compared with a sustainable one (Sitinplant MNGT). For this experiment were used micropropagated and inoculated (Glomus, Trichoderma and A. radiobacter) and not inoculated rootstocks produced in Greece during the acclimatisation phase and transferred to the nursery. Two additional treatments with Streptomyces lydicus WYEC 108 and Bacillus subtilis QST 713 were also applied. All trials were carried out in Bulgarian and Turkish experimental fields.
Results, obtained during last sampling in Bulgaria, showed that: i) rootstocks of GiseLa® 6 treated with Glomus gave the highest values of stem diameter and total dry matter compared to all other treatments. In addition, high stem diameter values were found in Trichoderma + Bacillus treatments; ii) Control Sitinplant treatment in OHF19-89 and Myrobolan 29C rootstocks showed the best values of stem diameter and accumulation of total dry matter. Moreover, in all the other biometric parameters measured the highest values were found with Trichoderma+Bacillus subtilis as well as Trichoderma+Streptomyces lydicus treatments; iii) for GF677 highest total dry matter resulted with Control Nursery treatment followed by the Glomus + A. radiobacter one. The analyses of plant mineral uptake were done in May, July and October 2009. Three plants (replications) were destroyed and analysed per treatment. Each plant was separated into root, stem and leaf parts. Each analysis was performed with 180 samples for mineral uptake analysing basic macro and microelements (N, P, K, Ca, Mg, Zn, Cu, Mn, Fe). The results obtained showed that the best uptake of macro elements was achieved by the rootstocks treated with microorganisms (Sitinplant management) compared to the plant control managed with conventional nursery techniques. Maximising the nutrient uptakes (N and K) by microorganism applications, we can reduce the risk of leaching losses of these elements into the ground water and so reduce the environmental impact. Groundwater pollution caused by agricultural activities is a serious problem in many regions of the world.
Analysis of persistence and presence of mycorrhizae. For each rootstock and treatment, five root samples were collected and used to estimate mycorrhizal colonisation (Glomus and Trichoderma). Arbuscular mycorrhizal colonisation analysis showed higher percentage of Glomus root colonisation in GiSeLa® 6 treated with Glomus. For GF677, Agrobacterium radiobacter was found in the plants that were inoculated with A. radiobacter combined with G. intraradices. Bacillus subtilis and Streptomyces lydicus, from the additional field treatments, were presented in sufficient quantity as it was expected. In particular, Bacillus subtilis was detected in GiSeLa® 6 and OHF19-89 rootstocks.
In Turkey, analysis of fresh and dry matter accumulation were carried out on ten plants (July 2009) for each rootstock and treatment, with a total about 190 samples examined. Determinations of macro and microelements were carried out on each rootstock treated in two separate samplings September and November (2009) with a total of 180 plants examined. In addition, specific mineral content analyses were carried out on each plant sample separating leaves, shoots and roots.
Inoculated roots were observed in all experimental plants denoting the success of the procedure.
Twenty plants per treatment and per rootstocks were selected randomly in 6 different sampling periods. On all collected plant samples, different biometric parameters were measured: plant height, number of leaves, stem diameter, number of lateral shoots and length of lateral shoots.
WP4. Optimisation of micrografting techniques on biotised rootstocks.
FITO SME produced micropropagated and biotised rootstocks to be used in grafting or micrografting tests and further experimentation. Plant material was transferred to the nurseries (Italy, Turkey and Bulgaria). All SMEs set up the grafting or micrografting method.
T.4.1 Optimum period for micrografting techniques.
To optimise and therefore improve the potential of the micro-grafting techniques, the SITINPLANT project will focus on the following experimental scientific issues:
i) optimisation of the micro grafting process on biotisated rootstocks with the aim of increasing the percentage of grafting success and plant quality;
ii) increase our understanding of the physiological and morpho-anatomical relationships of the grafting process in relation to biotisation;
iii) development of innovative techniques to reduce failures or delays in bud break in relation to biotisation.
The research compared the following possibilities:
i) micrografting in October-November, using actively-growing micropropagated rootstocks and buds of the cultivars to be grafted from mother plants in the same period;
ii) micrografting using buds of the cultivars to be grafted taken from mother plants in December-January and kept in a refrigerator and micrografted in February-April on actively-growing micropropagated rootstocks.
The research was carried out on the most commercially interesting fruit tree species (Apricot, Peach, Cherry, Pear), for each species using combinations of rootstock and scion to allow an accurate evaluation of the entire process. Plant material, micropropagated and treated with biological agents (see previous deliverables, about 2000 rootstocks) was transplanted to 0.9 L square pots with substrate mix TS1 (Klasmann-Deilmann Gmbh-Lithuania) at AGTE SME - Italy (July 2009). Buds were micrografted onto rootstocks in October 2009 and April 2010. Micrografting combinations: scions of Portici, Sunburst and Williams were supplied by the nursery "Iocoli" at Santarcangelo, Potenza-IT; Big Bang and TC Sun by the nursery "Vitroplant" at Cesena-IT. Preliminary trials indicate that "chip budding" is the best technique for obtaining higher grafting percentages also for reducing the scion material needed. To determine the most suitable time to perform micro-grafting, the rootstock Gf677 was grafted by chip budding in October using buds from scions of the same period. Also, in spring using buds from scions selected during winter and stored at 4°C.
Comparing the data obtained from the two grafting periods, the best performance was clearly shown to be in October. Autumn (October) is the most suitable period for micro-grafting, using mycorrhisated rootstocks, actively growing in pots under controlled conditions and fresh buds selected during the same week. The grafting efficiency is closely linked to cambial differentiation and callus formation. Unsuccessful micrograftings were shown to be associated with three main causes:
i) by the operator's technical skill in choosing the right material (diameter, quality, etc.);
ii) by the healing rate of the cut rootstock tissues and by the different growth rate between cultivar and rootstock meristems: when the rootstock is too vigorous, cultivar buds can be pushed out and die;
iii) by sanitary problems occurring when scions are stored in a refrigerator (mould formation).
T.4.2 Effect of biotisation of rootstocks on micrografting success.
This Task 4.2 aims to increase the grafting percentages in the micro-grafting technique using biotisated rootstocks. The related experiment evaluates the positive effects of biotisation on the micrografting success rate.
Grafting percentages. Comparing the data obtained from each rootstock/cultivar combination (micrografts done in October 2009), the micrografting technique and, in particular, chip budding were revealed to give good results in peach, 88% of total grafts took. Considering all combinations tested, the efficiency of the technique seems to be related to species sensitivity. Only GF 677 and peach showed interesting results. Unsuccessful micrograftings could be due to the healing speed of the cut rootstock tissues and by the different growth rates between cultivar and rootstock meristems.
Mycorrhization effects. Overall, rootstock mycorrhisation did not seem to affect the grafting potential of each species combination directly. There were no significant differences among treatments and controls on grafting success. The efficiency of micrografting is closely linked to cambial differentiation and callus formation.
T.4.3 Effect of biotisation on grafting success of the plants in the nursery.
Mycorrhizal fungi have been shown to increase uptake of water and mineral nutrients by plants. Until now, there have been no clear relationships between mycorrhizal inoculation and grafting/budding success. The objective of this research was to record the effect of biotisation on grafting success of the plants in the nursery.
All experiments were carried out in Turkish and Bulgarian field sites to study the following topics:
i) effects of biotisation on grafting success rates;
ii) influences of biotisation on physiology of grafted plants.
All analyses were carried out under one or other of two different field management systems:
i) conventional nursery management;
ii) sustainable Sitinplant management.
GiSeLa®6 rootstock grafted with cherry cultivar Van: the best rates were generally found with the Trichoderma biotisated plants which exceeded the controls by between 3% and 11%. OHF19-89 rootstock grafted with the pear cultivar William: compared with the results obtained for the other experimental rootstocks, the best rates of up to 60%, were achieved with the Glomus + Trichoderma combination. As with the previous rootstock, there were no significant differences for the additional bacterial treatments. Myrobolan 29C rootstock grafted with the plum cultivar Angeleno: the best rate was for the Glomus + Trichoderma variant. GF 677 rootstock grafted with the peach variety Royal Glory: there were no significant differences between mycorrhizated and non-mycorrhizated variants in the SITINPLANT plots.
Project experimental field - Bursa, Turkey. The micropropagated material obtained from Fitotechniki (Greek-SME partner of the SITINPLANT) was planted on 26 June following standard procedures, after agro technical soil cultivation and setting up for drip irrigation. Project treatments were compared to classical nursery applications. Two antagonistic microorganisms (Bacillus subtilis and Streptomyces lydicus) were used. The number of plants used in the trial at Bursa was about 13500.
Vegetative buds were T-grafted onto rootstocks treated with biological agents in September-October 2009.
The percentages of successfully grafted plants (shooting) were observed and calculated for each variety in a 4x3 table (levels of mycorrhizae and bacteria). Chi-square tests were performed on observed and expected numbers of grafting results in three different ways:
1) bacterial treatments were combined for each mycorrhizae treatment; and mychoryzae treatments versus grafting results (success/failure);
2) mycorrhizae treatments were combined for each bacteria treatment; and bacteria treatments versus grafting results (success/failure);
3) bacteria-mychoryzae combination treatments versus grafting results (success/failure).
WP5. Optimisation of growing techniques.
The general objective of this WP5 was to define the correct nursery techniques management that are compatible with microorganism symbiosis and that ensure the most favourable conditions to obtain good quality of micrografted plants in pots and grafted plants in the nursery.
At this stage plant material was prepared with about 975 rootstocks micro-grafted in Italian nurseries, 7879 in Bulgaria and 7894 in Turkey.
The WP5 aimed at studying:
a) persistence of microorganisms on micrografted plants in pots (T.5.1);
b) effects of different managements in the nursery on persistence of microorganisms of grafted plants on inoculated and non inoculated rootstocks (T.5.2);
c) evaluation of the growth and quality of micrografted plants grown in pots and grafted plants in the nursery (T.5.3).
T.5.1 Persistence of microorganisms on micrografted plants grown in pots.
UBAS conducted trials under controlled pot conditions according to the existing AGTE SME production processes. The first step was to check for microorganism persistence. Plant material, micropropagated and treated with biological agents was transplanted into 0.9 L square pots with substrate mix TS1 (Klasmann-Deilmann Gmbh-Lithuania) at AGTE SME - Italy (July 2009). Buds were micrografted onto rootstocks in October 2009 and April 2010. Plants were grown under 50% shade net. Water was automatically distributed by overhead mini-sprinklers (range 2.5 m, 120 L/h) with three applications during July and August and two in September and October 2009. Fungicides were not used to avoid detrimental effects on the biocontrol agents. Rootstocks were treated with Actara (Thiamethoxam), 40 g/hL; grafted plants with Confidor (Imidacloprid), 50 ml/hL to control aphid infestations. A basic Scotts (20-20-20 + micro), 0.5 kg/hL and potassium sulfate 1 kg/hL were applied twice by fertigation in September 2009 before grafting. Different foliar fertilisers 31-11-11, 330 g/hL plus 330 ml/hL of organic N 5%, and 18-6-2+12, 0.186 g/pot were applied respectively in April and three times in May 2010. Chip budding was done as described.
Presence and persistence of microorganisms. The persistence of bio-control agents was checked on 10 plants per treatment, at the end of the production cycle in June 2010. Qualitative and quantitative measurements were carried out on roots: infection and arbuscular percentages, intensity of colonisation for Glomus; and on substrates for CFU Trichoderma detection.
There were no significant effects of mycorrhization on graft take.
Glomus intraradices. Considering the frequency of system mycorrhisation (F%), treated plants showed the best results, 100%, in all four rootstocks, compared to controls. Controls were probably infected by natural agents always present in the soil or in the irrigation water. For all treated plants, colonisation intensity (M%), the most important parameter to evaluate the colonisation, ranged between 40 and 85%, reaching the highest values in micrografted Gf677, compared to controls; the differences were statistically significant. The arbuscular presence (a% and A%) was evident in Glomus treated Gf677, about 50%, in Myrobolan 29C plants, 10%, and in OHF19-89, about 20%. Arbuscular differences were, however, less marked, above all in GiSeLa®6.
T.5.2 Effect of different managements in the nursery on persistence of microorganisms of grafted plants on inoculated and non inoculated rootstocks.
AUTH partner monitored the persistence in the field under nursery management, or modified Sitinplant management of microorganisms used to inoculate plants at the rooting phase and again at transplanting in the Bulgarian and Turkish experimental fields. FITO SME produced micropropagated and biotised rootstocks to be used in grafting tests and further experimentation. Plant material was transferred to the nurseries (Turkey and Bulgaria). All SMEs set up the grafting and management methods. Soil and root samples were collected from the rhizosphere of the field plants. Analysis for arbuscular mycorrhizal fungal root colonisation from the Turkish samples was conducted in Turkey.
Trichoderma harzianum. For T. harzianum two different media were used. Malt extract agar is non-specific to Trichoderma sp, and we have previously used it for detection of T. harzianum, however its lack of selectivity made quantification difficult: other fungi would also grow on this medium and as a result only qualitative data were possible (presence/absence), dividing the number of positive plates with the total number of plates/replicates. For quantitative data a semi-selective medium was employed, however, until sporulation (required for positive identification) the fungus may also spread on the plate and colonies may overlap, therefore on one occasion qualitative rather than quantitative data were reported with this medium also. We must note that almost no Trichoderma was recorded in both control treatments and media used, particularly in the nursery management plots. Initially, there was nothing detected in Sliven (Bulgaria) in October 2009 with the semi-selective medium, and numbers remained low in the Bursa (Turkey) samples of December 2009. However, the Trichoderma population increased in 2010 and peaked in October 2010 Sliven rootstock rhizosphere. Although there was high variability in the results, and no discernable pattern regarding interaction of T. harzianum with the other organisms applied, and taking into account results from both media, the form of the inoculum does not seem to make a difference to T. harzianum persistence. However, there was some indication from Myrobolan 29C and the semi-selective medium that Trianum P gave slightly higher numbers than Trianum-G after re-application. In addition, due to data variability, and appearance and disappearance of the organism with time with no perceptible pattern, re-application, regardless of inoculum form, does not seem to offer any particular benefit to persistence of T. harzianum.
The molecular identification data confirmed that most of the isolates were Trichoderma harzianum T22. In Bulgaria all (100%) of the isolates were T. harzianum T22, and in Turkey (62%) of the isolates, with local Trichoderma sp. also being present. On the basis of their morphological characteristics or due to molecular analysis of the internal transcribed spacer 1 and 2 regions (ITS1 and ITS2), Trichoderma virens (25% of the isolates obtained) and Trichoderma spirale (13% of the isolates obtained) were also present.
T.5.3 Evaluation of the growth and quality of micrografted and grafted plants.
Evaluation of the growth and quality of micrografted plants grown in pots.
UBAS conducted trials under controlled pot conditions according to the existing AGTE SME production process. In particular, UBAS studied:
a) effects of symbiosis on micrografted plant growth and quality:
analysis of dry weight of micrografted and grafted rootstock roots system; plant growth; analysis of dry matter accumulation and partitioning between different plant organs; analysis of total mineral elements uptake of micrografted rootstocks in pots;
b) effects of symbiosis on plant water uptake and root conductivity.
Effects of symbiosis on micrografted plant growth and quality. Dry weight of micrografted plants at the end of the production cycle (plants ready for the market). Glomus and Trichoderma biotised plants reached total mean dry weights of 1.46 and 2.21 g greater than the controls (higher by 19.6 and 29.8% respectively). Differences between the two treatments were not significant; but it is necessary to stress that, although there was a high variability for each treatment, plants inoculated with Trichoderma grew on average about 1 g more than the Glomus ones (8.5% more). The plants of Big bang/GF677 and TC Sun/Myr. 29C combinations reached a dry weight of 11.96 and 10.58 g, and had diameters of 4.57 and 4.66 mm, respectively; while the Sumbuster/GiSeLa®6 and Williams/OHF 19-89 combinations grew less, showing mean values of total dry weight of 7.46 and 4.58 g respectively. The growth ratio between the scion and rootstock of the different combinations emphasised the significant differences. In particular, while for the combination Big bang/GF677 the dry weight ratio was 0.97 i.e. there was a perfect linear growth rate of bionts, for the other three combinations was clear the predominance of the rootstock, with a greater growth of 1.23 1.76 and 2.30 g, respectively in Williams/OHF19-89, TC Sun/Myr. 29C and Sunbuster/GiSeLa®6.
The analyses of dry weight data for each combination confirms less control growth compared to inoculated plants; except for the combination William/OHF19-89, in which difference were not significant. There was a mean growth of about 10% greater in Glomus plants for the Big bang/GF677 combination, while, for the other combinations, Trichoderma treatment induced the best results (plants were about 20% bigger than the controls).
Evaluation of growth and quality of grafted plants in nursery.
The most important step for practical implementation of micropropagated, inoculated and grafted rootstocks in the nursery was a complex study of the interaction between mycorrhizae and antagonist biocontrol micro-organisms on plant growth and quality, as well as on the efficiency of nutrient uptake and disease incidence during the whole vegetation period. At the same way, the types of grafting as well as grafting compatibility, between rootstock and scion, were considered. Environmental conditions during the experimental period had a big influence on the above mentioned complex of interactions.
In vitro micropropagated and biotisated rootstocks were obtained from SME Fitotechniki - Greece. They were planted in the Bulgarian project experimental field (Sliven region) on 16 April 2009. The rootstocks were grafted with the cultivar scions on 14-15 and on 16 August 2009 by T-budding, 10 cm from the soil. In Turkey the planting was performed on 26 June 2009 by standard procedure, after the agrotechnical soil cultivation as well as the construction for drip irrigation.
The following experiments were carried out with successfully grafted plants of rootstock/scion combinations. Distribution of experimental plants was done on randomised plots, related to the project experimental plan for 2010, including additional mycorrhizal and bacterial treatments with the same bio-agents in some of the GF677/Royal Glory combinations. Additional treatments were done inside the experimental plots with the same mycorrhizal inoculi used in the first stages of in vitro rootstocks production in Fitotechniki, Greece. Regarding the positive results obtained in 2009 with Trichoderma/Baccillus subtilis and Trichoderma/Streptomyces lydicus treatments, both Trichoderma inoculations were also included as different combinations. All microorganisms were applied manually, as close to the roots as possible. On the basis of previous results, the amount of nitrogen and potassium was increased by 30%. The fertilisers in the SITINPLANT plots were applied proportionally by fertigation (approx. 2 times per month, depending on rainfall), altogether 12 times during the whole vegetative period. The distributions of fertilisers in the conventional SME plots were done proportionally twice during the vegetative period by soil implementation and leaf feeding. Growing parameters were measured: height (rootstock upper part + scion) and stem diameter (scion, 10 cm from the grafting point). The analyses of dry matter accumulation were done at the start and at the end of the plant vegetation cycle, during the months June and October respectively. Three plants were destroyed and analysed per treatment. Each plant was separated into four parts: rootstock root, rootstock upper part, variety shoot, variety leaves. Fresh/dry matter accumulation analyses were done for each plant part. The analyses of mineral uptake were done twice in the vegetation period (June and October 2010). Each sample was checked for mineral uptake of the 6 basic macro and microelements (N, P, K, Ca, Mg and Fe).
Phytonematodes. After two years of experiments, mycorrhizae contrasted with pathogen development; treatments showed better results than the controls with a reduction in the number of ectoparasite phytonematodes under nursery conditions. Moreover, the density of semi-parasite and free living ones was maintained. It means mycorrhizal treatments can be strongly recommended as a tool for biological control.
Biometric analyses. Results obtained after the second vegetative period can be summarised as:
i) Glomus treatment appeared to induce the best growth for GiSeLa®6/Van experimental plants;
ii) Trichoderma experimental plants showed best height results in OHF19-89/William rootstock/scion combination. All variants demonstrated aligned and good plant growth with regard to stem diameter but without significant differences;
iii) Glomus+Trichoderma variant induced the best height growth rate in both the controls with 12% (Control SITINPLANT) and 17% (Control Nursery) in the Myrobolan29C/Angeleno combination. No remarkable differences were measured in stem diameter among all the experimental plots;
iv) additional treated variants Glomus (AG) and Glomus+Agrobacterium (AG) provided best final results in GF677/Royal Glory experimental plots.
Fresh/dry matter accumulation. The GiSeLa®6/Van: Glomus variant clearly had the best fresh and dry matter accumulations as well as FW/DW ratios. OHF19-89/William: in this case, the Trichoderma variant showed the highest fresh and dry matter accumulation. Myrobolan29C/Angeleno: Glomus+Trichoderma and Glomus variants provided the highest fresh and dry matter values and fresh/dry matter ratios. GF677/Royal Glory: Glomus treatments influenced the fresh and dry matter accumulation as well as FW/DW ratios. The positive effects of additional field mycorrhisation were confirmed.
Basic mineral elements uptake. GiSeLa®6/Van: both Glomus and Trichoderma variants showed the best mineral uptake rates, where Glomus clearly exceeded in N and Fe accumulations and Trichoderma in Mg. Concerning K and Ca mineral elements Glomus and Trichoderma experimental plants showed very close accumulation rates. OHF19-89/William: in all cases the Trichoderma variant was demonstrated to have very good accumulation activity. Myrobolan29C/Angeleno: Glomus+Trichoderma for the rootstock part and Glomus for the scion part presented the best final absolute values. GF677/Royal Glory: Glomus+Agrobacterium induced the best results. Additional treatments gave an advantage in all cases.
WP6. Definition and evaluation of innovated process.
The main outcomes are working protocols and specific technical procedures containing information related to the phases of the optimised cycle and outlining possible critical points.
T.6.1 Production scheme of micropropagated and biotisated rootstocks.
In advanced agricultural systems, grower performance is closely tied up with nursery propagation efficiency. To respond to modern plant production requirements, nurseries must combine economic and environmental aspects. New process knowledge is important to define the innovated plant production describing the criteria for optimising micropropagation and biotisation methods in rootstock production and streamlining nursery organisation.
The company Fitotechniki (FITO), in collaboration with AUTH, had the responsibility in the Project of developing the protocol for production of the inoculated and micropropagated rootstocks, estimating the qualitative, environmental and economic benefits.
The inoculation by dipping, on the basis of experiments carried out, was more efficient. Whereas the possibility to propagate fresh culture of T. harzianum, results will be better. Trichoderma is an excellent root and peat coloniser resulting in promotion of specific plant growth characteristic and suppression of P. ultimum and R. solani mediated disease.
T.6.2 Production scheme of micrografted and grafted fruit tree plants using inoculated micropropagated rootstocks grown in pots and in the nursery.
The protocol for fruit tree production was developed by the Italian, Bulgarian and Turkish partners of the Project, in collaboration SME-RTD, estimating the qualitative, environmental and economic benefits gained.
Micrografted fruit trees. A new technical protocol was defined at the end of the Italian trials as a result of the two-year experimental activity. The production scheme is developed for the rootstocks GiSeLa®6, OHF19-89, Myrobolan29C and GF677. Scions Portici, Sunburst and Williams were supplied by the nursery "Iocoli" at Santarcangelo, Potenza-IT; Big Bang® and TC Sun by the nursery "Vitroplant" at Cesena-IT.
Rootstocks are obtained by micropropagation and delivered to the nursery at the end of the summer (height about 15-20 cm, diameter 2-3 mm). Plants in the nursery are transplanted in square pots (size: 9x9 cm, 13 cm height) that avoid root circling and maintain best growth to the end of the process, adding white peat and a complex slow-release fertiliser. It is important that the mix is poor in phosphorus and that this element is not applied during regular fertilisations or it could interfere with the symbiotic fungi. In the same way, systemic fungicide use should be avoided. For the micrografting, the "chip-budding" technique is used; it should be carried out in the period September-October. Production efficiency defined grafts 50-60 plants/hour/person.
The few days following the grafting are extremely tricky, in particular it is important to assure temperatures of 25-28°C and RH of 80-90%. Plants are maintained in a protected environment and under optimal water/nutritional/sanitary conditions. In April plants are ready for the market, potted plants with a height of about 40-50 cm and a diameter of 4.5-5 mm. The cycle previously proposed was best in terms of plant quality and efficiency in grafting take. But it is possible to develop different processes. These alternative cycles can apply to specific commercial needs. It is possible to use stored buds (sampled in the previous year), reducing the production cycle, from 9 months of the first proposed to 4-5 months. The production cycle under greenhouse conditions is characterised by high flexibility and may be started at any time of year.
Grafted fruit tree plants. The production scheme presented is based on the results from two-year experiments carried out in the Bulgarian and Turkish project experimental fields. It could be implemented in other environmentally suitable regions in Bulgaria and Turkey with some adjustment, mainly in the fertilisation plan, depending on preliminary soil and water analyses. The production scheme is developed for the rootstocks GiSeLa®6, OHF19-89, Myrobolan29C and GF677. The scion varieties grafted on them in Bulgaria were respectively cv. Van (cherry), cv. William (pear), cv. Angeleno (plum) and Royal Glory (peach), in Turkey cv. Skeena, cv. Black Diamond and cv.s Angeleno, Royal Lee and Tardired, for the SME applications, GF677 was grafted with the cultivars Tardired and Royal Lee. The rootstock should be virus-free, in vitro obtained and treated twice with Trichoderma harzianum (T22 strain) and Glomus intraradices (separate and in combinations) at the beginning of the acclimatisation stage and before transplanting in the nursery.
Potential Impact:
The project success will allow production of micrografted and grafted plants using inoculated micropropagated rootstocks. The following main benefits are achieved:
-Improvement of plant tolerance to biotic and abiotic stress through the establishment of a long-lasting plant symbiont colonisation of root and leaf surfaces;
-Reduction of chemical pesticides used in the nursery and orchard through the induction of a systemic resistance;
-Reduction of the use of chemical fertilisers by stimulating root development and increasing uptake efficiency through natural agents;
-Reduction of agricultural environmental impact by treating unhealthy soils through inoculation of plant symbionts which decrease the activity of deleterious soil-born microflora and inactivate toxic compounds in the root zone;
-Reduction of costs and time of the nursery production cycle.
Production scheme of micropropagated and biotisated rootstocks.
As a result of the new production scheme plants are healthier, and there are very few losses, due to more efficient suppression of soil pathogens, in the acclimatisation phase. In fact, previously, using fungicides, the company FITO was losing about 10% of the plants during the acclimatisation phase while now, with the application of antagonists, only a small percentage (approximately 3%) is lost. In addition, plants are more homogeneous in development, and there is a shortening of the hardening period by one week, which translates to worthwhile savings in personnel and greenhouse space. With the previous production scheme pesticides were used pre-emotively, but are now not included in the current scheme: this is an enormous benefit for the personnel, the SME and the environment. Previously the personnel had to use the pesticides, and re-entrance to the greenhouses was forbidden for some time after application. Now exposure to chemicals and the associated health effect risks are minimised and staff feel safer in a healthier environment. Environmentally, the risk of contaminating the soil and the groundwater with chemicals is minimised. There is also a financial benefit. The cost of pesticides with the previous scheme was 5500 Euros per million micropropagated plants produced (current production of the SME is about ten million plants per year), while the cost of applying and using the microorganisms for the same number of plants is 1400 Euros (using commercial inoculum).
Production scheme of micrografted fruit trees in pots.
To estimate the impact of the project results application, not only should the quantitative cost reduction of the overall production be taken into account but also the improvements in plant quality using environmentally friendly techniques that SITINPLANT has achieved. The overall production cost reduction is estimated at for 35% for bi-member plants of 30-40 cm height and of about the 25% for producing micrografted plants of 80-120 cm height. The project results, with further RTD activities, can also be transferred to other sectors such as the forest sector, in particular for the recovery of areas destroyed by fire or natural calamities and for sectors such as specialised greenhouse floriculture. Moreover, all sectors related to agriculture, such as food industry, will be positively affected by the project results indirectly. Another relevant aspect of this project is the environmental impact, since the inoculated and micrografted plants reduce the input of chemical fertilisers and pesticides, reducing soil and water pollution.
Production scheme of grafted fruit tree plants.
Traditional grafting processes are characterised by variability, low scion affinity, poor plant development, low quality, susceptibility to pathogens, non-certified plants etc. SITINPLANT management provides a competitive price, the overall production cost reduction is estimated at 30% of the traditional cost, in addition to the listed advantages in terms of innovation and quality plant production. Other main outcomes and benefits for SME in Bulgaria and Turkey are that SITINPLANT management allows a drastic reduction of the application of N and K by 71% and 86% respectively and 100% for P in the first year. In the second year nursery cycle, reduction percentages are respectively 41%, 56% and again 100% for P. Soil-born pathogen control was found to be possible through inoculation with antagonist microorganisms Baccilus subtilis and Streptomyces lydicus which is an enormous advantage from an economic and environment point of view. The priority use of biological technologies will facilitate and reduce the time for the transition from conventional to organic farming for SMEs. In addition, implementation of the SITINPLANT management production scheme could improve the social status of the people living in Bulgaria and Turkey affected regions.
A detailed IPR and knowledge management plan was worked out where the IPR on the different project results was defined. The IPR and knowledge management addresses the following issues:
-ownership of the project results (foreground knowledge);
-licensing of pre-existing know-how;
-transfer of the knowledge gained within the project;
-confidentiality of project results and dissemination strategy.
This is due to the following consideration:
-It is very difficult to differentiate the use of such knowledge among the different companies;
-SMEs involved are not competitors (they operate in very different markets);
-The level of investment for each SME is considered fair for the market in which it operates and for its specific expected return.
Specific agreement on Intellectual Property was undertaken with the RTD performers. This was based mainly on the following rules:
-RTD providers are able to realise publications and present the results obtained during the project in relevant international conferences but the information to be provided should not be sensitive information.
-The results of the project are property of the SMEs involved. The handling of the Intellectual Property Rights was defined through the Consortium Agreement that proposers created and signed before the end of the first month of project activity.
The objectives of the Dissemination phase are:
i) to disseminate information about the project, its objectives, its approaches and its results;
ii) to facilitate collaboration and information exchange between relevant research and agricultural communities;
iii) where applicable to promote the use of techniques resulting from the project amongst the target groups: research entities, SITINPLANT stakeholders, others;
iv) to create two-way communication channels with stakeholders, research communities and industry for disseminating the project deliverables and conclusions;
v) to ensure that the products of the project live on in a commercial context, this way assisting the Exploitation of the project results.
Conclusions
Impact for the SME participants
The project success has clearly improved SMEs competitiveness as far as project results which were targeted mainly to the nursery plant sector - the core business of all SMEs proposers.
Several advantages, in terms of increased competitiveness are foreseen for SMEs proposers:
-Reduction of production cycle times: from 2 years to 3-8 months.
-Increased tolerance of the plants produced: with the new techniques plants will be more tolerant to pathogens and will make more efficient use of the mineral and water resources of the soil.
-Reduced production costs: through the use of biotised and micrografted plants.
-Increased know how provides several direct and indirect commercial benefits, also taking into consideration consulting services that can be offered to implement the innovative techniques.
Improvement of industrial competitiveness across the European Union
The plant nursery sector plays a very important role in the wider agriculture sector, since quantity and quality of yield depend mostly on the quality of the plant material used for orchard plantation. This sector, as well as the wider agriculture sector in Europe is currently experiencing competitiveness problems due to the emergence of global competitors able to offer aggressive commercial policies. For this reason it is essential for European SMEs to innovate their production processes to remain competitive and to comply with environmental policies.
Employment opportunities
The increased competitiveness of SMEs in a growing market such as fruit tree nursery sector (about 30%) produces new employment opportunities (20-30 new workers, above all women, during the project).
European Cohesion
The project proposes a strong collaboration among partners coming from four European countries, thus improving European cohesion.
List of Websites:
http://www.unibas.it/sitinplant/home.htm
The SITINPLANT project, funded by the European Commission under the 7th Framework Programme, started officially on 1 September 2008.
The aim of the SITINPLANT project was:
- to use mycorrhizae, antagonist bio-control microorganisms, during the acclimation phase of micropropagated rootstocks and in the nursery,
- to optimise an innovative micrografting technique.
During the two years of activity, the work achieved the following main objectives:
- Optimisation of the inoculation method on micropropagated rootstocks;
- Development of the correct interaction between mycorrhizae and antagonist biocontrol micro-organisms on controlling disease incidence and improving plant nutrient uptake;
- Optimisation of micrografting techniques;
- Optimisation of nursery growing techniques for micrografted and mycorrizated plants;
- Implementation and testing of the innovated plant materials under a range of pedo-climatic conditions.
Project Context and Objectives:
European and worldwide policies are increasingly working to limit the use of chemical substances for fertilisation and plant protection so as to reduce their negative environmental impacts and to increase soil fertility. In the plant nursery sector there is widespread use of chemical pesticides to repress plant pathogens and of synthetic fertilisers to stimulate plant growth. Both practices lead to a reduction in the complexity of the soil microflora and to a decline in overall plant quality. As a result, innovative nursery plant production processes are urgently needed to produce plants that are more tolerant of soil-borne pathogens and also more efficient in using the natural soil resources (minerals, water, etc.) in all phases of cultivation (in the tissue-culture laboratory for rootstock production, the nursery and the orchard).
During the two years of activity the work completed the following tasks:
T.2.1. Studies on physiological and molecular relationships between symbiont micro-organisms and plant root systems during the acclimatisation phase and in the nursery;
T.2.2. Effect of agronomic and environmental parameters on the establishment and continuance of symbiosis in plants grown in pots;
T.2.3. Plant-soil-microbe interactions in the rhizopshere influencing the rate of inoculation and efficiency of biocontrol;
T.3.1. Optimisation of techniques to inoculate micropropagated rootstocks at different growth stages;
T.3.2. Analysis of symbiotic effects on plant growth and quality in the nursery;
T.4.1. Definition of the optimum period for micrografting techniques in relation to biotisation;
T.4.2. Effects of biotisation of the rootstocks on micrografting success;
T.4.3. Effects of biotisation on grafting success of the plants grafted in the nursery;
T.5.1. Persistence of microorganisms in micrografted plants grown in pots;
T.5.2. Effect of different managements in the nursery on persistence of microorganisms in grafted plants on inoculated and non inoculated micropropagated rootstocks;
T.5.3. Evaluation of the growth and quality of micrografted and grafted plants on inoculated and non inoculated micropropagated rootstocks;
T.6.1. Production scheme of micropropagated and biotisated rootstocks;
T.6.2. Production scheme of micrografted and grafted fruit tree plants using inoculated micropropagated rootstocks grown in pots and in nursery;
T.7.1. Plan for use and dissemination of the foreground;
T.7.2. Project website.
Project Results:
WP2. Interaction of mycorrhizae and antagonist biocontrol micro-organisms.
The aim of this WP2 was to study the relationships between symbiont microorganisms and rootstock systems and to define the optimal agronomic and environmental conditions that foster the establishment and the continuance of symbiosis.
T.2.1 Study on the physiological and molecular relationships between symbiont microorganisms and the plant root system during the acclimatisation phase and in the nursery.
Part of the work in the T.2.1 was completed during acclimatisation. In particular, the results achieved in the first year showed that inoculation of the substrate with Trichoderma and Glomus of micropropagated rootstocks significantly decreases the percentage of dead plants during the acclimatisation phase. Inoculated plants without fungicide treatments reduced plant mortality. Because these results represent very important achievements, SME has already changed the plant production process reducing or eliminating the use of fungicides during the acclimatisation phase.
The activity carried out focused on: studies of gene expression and protein characterisation during the interaction between Trichoderma and plant roots in vitro. Trichoderma harzianum strain T-22 controls many plant pathogens through mycoparasitic interactions with the target fungal organism and it is believed that extracellular cell wall degrading enzymes produced by the antagonist play an important role in these interactions. It has also been reported that T. harzianum has the ability to directly enhance root growth and plant development in the absence of pathogens and it has been suggested that this is due to the production of a growth-regulating factor by the fungus. In spite of their theoretical and practical importance, the mechanisms responsible for the growth response due to the direct action of T. harzianum on plants have not been investigated extensively. All the above-cited observations indicate the versatility through which T. harzianum can manifest biological control activity. The application of T22 to in vitro cultured GiSeLa6® rootstocks (Prunus cerasus x P. canescens) during the rooting phase resulted in greater shoot lengths, numbers of leaves, numbers of roots and stem diameters. For this reason, we continued the experimentation on this rootstock, trying to explain the biochemical basis of plant-growth-promoting activity of T22. On the basis of the results of the previous experiments and of other research (RIF), we believe that the higher shoot and root growth in the plants inoculated with T22 could be due to a different phyto-hormonal balance or to medium acidification and redox fungal activity.
Hormonal regulation may be achieved by:
(1) a balance or ratio between hormones,
(2) opposing effects between hormones,
(3) alterations of the effective concentration of one hormone by another and
(4) sequential actions of different hormones.
For this reason not only is the hormone level very important per se but also their ratios. Considering the positive effects on shoot and root growth in plants inoculated with T22, we supposed that changes in auxin/cytokinins levels could be involved. It is known that a higher auxin to cytokinin ratio promotes root formation, whereas a higher cytokinin to auxin ratio promotes shoot-bud formation. Several lines of evidence indicate that while auxins stimulate root formation, cytokinins inhibit it.
Firstly, the results obtained demonstrate that the extraction methods, particularly complicated for phyto-hormones, were successful. In particular, two points during the extraction procedures are considered critical: maintenance of temperatures lower than 4°C during all the extraction steps and the concentration of the solvent mixture (not completely dry) with nitrogen flow to avoid the oxidation of hormones. For this latter point, a particular method using a nitrogen evaporator was designed and this allowed desiccation of all samples together and relatively fast. The competitive ELISA assay allowed a quantitative profile of the three hormones examined. In particular, the method discriminated well between IAA and TR-ZEA, whereas DH-ZEA was not present in either leaves or roots. This is normal as DH-ZEA is a precursor of TR-ZEA in the biosynthetic pattern used as 'zeatin storage' by plant cells. Thus, its absence suggests that the precursor TR-ZEA was not present before T22 inoculation and that all the TR-ZEA detected was produced ex novo after T22 exposure. The levels of IAA increased significantly after inoculation with T22 in both leaves and roots. This could explain the greater root and shoot growth in inoculated plants. Generally, IAA was higher in leaves but this is normal as it is produced mainly in leaves and then transported to the roots. As IAA regulates lateral foliar bud formation, it is directly related to the number of leaves. We have already demonstrated leaf number to be higher in T22-inoculated plants.
T.2.2 Effect of agronomic and environmental parameters on the establishment and continuance of symbiosis in plants grown in pots.
The experiments were carried out to evaluate during the growth cycle under pot conditions: a) the presence and persistence of microorganisms; b) the effects on persistence of agronomic parameters (inoculation method, peat type, pot shape, fungicide application); c) the effects of symbiosis on plant root conductivity; d) the effects of symbiosis and agronomic parameters on plant growth and mineral partitioning.
Two sets of plants produced in Greece were used for the Italian experiments:
- the first set was used to analyse microorganism persistence, effects of different substrates and pot shapes, and root conductivity. In particular, the rootstocks before the first acclimatisation phase (October 2008) were inoculated by mixing the substrate with microorganisms. The plants were then re-inoculated by water solution just before transfer to the nursery. All material was kept under greenhouse conditions of the SME Fitotechniki until March 2009 when it was transferred to Italy for further studies.
- the second set of plants was used to study the effects of inoculation methods and fungicide application. Those plants were acclimatisated in February 2009 (second acclimatisation period). Before the acclimatisation phase plants were inoculated with microorganisms by mixing into the substrate or by dipping in a water suspension. All plants were kept under regular greenhouse conditions of the SME Fitotechniki, including fungicide application until the transfer to Italy.
Concerning the first point (a), in Italy in pots the presence and persistence of biocontrol agents were checked on 10 plants per treatment and on three different sampling periods (April-July-December 2009). Qualitative and quantitative measurements were carried out on roots: infection and arbuscular percentage and intensity of colonisation for Glomus (590 samples-Biologic units); and on substrates: Colony Forming Units (CFU) for Trichoderma (680 samples-biologic units). Results showed that Glomus intraradices colonisation intensity for all rootstocks reached the highest values in July (2009) from 50 to 80%, the intensity percentages decreased successively in December (2009) as in the first measurement (April 2009). Glomus + Trichoderma (in Myrobolan 29C, GiSeLa®6 and OHF 19-89) or Glomus + Radiobacter (in GF677) treatments showed the highest colonisation intensities. There were significant differences in arbuscular abundance between control and treated roots, the values ranged between 0-10% and 10-60%, respectively. Trichoderma CFUs were higher in Myrobolan 29C and GiSeLa®6 rootstocks at the second sampling period (July) with values of 3-5 x 104 CFU x g-1. Glomus values reduced to 1 x 104 CFU x g-1in December 2009. In GF and OHF19-89, CFUs were fewer than previous rootstocks but remained constant or increased until December. Therefore, the general colonisation trend of Glomus intraradices and Trichoderma harzianum showed a peak in July followed by a decrease in December.
(b) Effect of inoculation methods: inoculum mixed into the peat substrate or dipping the plants in the water solution were evaluated for CFU of Trichoderma in GF677 rootstocks. In December, CFUs calculated for the dipping method increased to 2.5 x 104 CFU x g-1. This value was significantly higher than values obtained in the mixing method (1 x 104 CFU x g-1). Evaluation the effects of the two substrates types, enriched (TN) and impoverished (TP), were compared for mycorrhization rate and the persistence of Glomus and also on CFU capacity of Trichoderma in all rootstocks. Differences induced by the two substrates were not significant. The effects of two pot shape, round (VT) and square (VQ), were compared on mycorrhization rate and persistence of Glomus intraradices, in all rootstocks. Round pots enhanced root mycorrhization: intensity percentages were about 50% higher and arbuscular abundance x4 fold higher than in square pots, in July. Values decreased for round pots and differences became non significant in December. Colonisation for square pots remained lower, instead increased by about 10% from July to December. Glomus intraradices showed better colonisation in non fungicide treated plants, during the period April - July. Effect of the fungicide applications were evaluated on mycorrhization and persistence parameters of Glomus intraradices and on CFU capacity of Trichoderma for GF677 rootstocks during the period April-December 2009. Plants treated with fungicides showed, as for Glomus, CFUs of Trichoderma constant and significant higher of 30% in December. Microorganisms in plants without fungicide application developed better during the first phases of their colonisation. In addition in plants treated with Glomus intraradices and Trichoderma harzianum effects of symbiosis on plant root conductivity (Lp) showed an increase in Lp of about 30% above the controls. Further studies on the effect of symbiosis on plant growth parameters were carried out on 620 plants in the first and second samplings and on 80 plants in December. Results showed that: Glomus+Tricoderma (G+T) and Glomus+Radiobacter for GF677 (G+R) treatments enhanced plant growth by about 30 and 40% above the controls respectively. Positive results were also obtained using enriched substrates, in this case plant growth doubled.
T.2.3 Plant-soil-microbe interaction in the rhizosphere influencing the rate of inoculation and the efficiency of biocontrol.
The general objective of task 2.3 was to study the factors involved in the plant-soil-microbe relationship that increase the efficiency of biocontrol, in particular to evaluate the capability of biological agents against the most important fungal pathogens and the strategies to enhance these biocontrol activities.
UBAS and AUTH main activities were focused on: a) studies on the identification and characterisation of secondary metabolites involved in the bioactivity of Trichoderma; b) Trichoderma biocontrol efficiency against fungal pathogens (Fusarium, Phytium and Rhizoctonia).
Some Trichoderma spp. are considered to be important biocontrol agents against plant pathogen fungi since they use various defense mechanisms such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. Trichoderma harzianum is often used in liquid or dry formulations of bio-pesticides, bio-fungicides and bio-stimulants. These species also have the ability to produce volatile antifungal compounds that have a partial role in the inhibition of growth of plant pathogen fungi. In this part of the study we investigated the growth inhibition of three target fungi (Fusarium oxysporum, Pythium ultimum and Rhizoctonia solani) due to the production of volatile compounds by Trichoderma harzianum T22 and Trichoderma asperellum B1.
Potato dextrose agar (PDA) (20 mL) was poured onto Petri dishes. A 5 mm diameter agar disc was excised from the leading edge of a two-day-old pure culture of T. harzianum T22 and T. asperellum B1 cultures was placed at the centre of each agar plate. Next, a disc of the same size was taken from each target culture and likewise placed on another agar plate. The lids were removed, and target culture plates were immediately placed over each of the Trichoderma plates and held in place with adhesive tape. The head space prevented any physical contact between the pathogens and antagonistic fungi, so that the volatile compounds were formed and confined to the interior atmosphere of the two plates. For the control plate, only the targets were cultured on each Petri dish. The plates were randomised and incubated at 28±2ºC for 4 days. The diameters of target colony cultures were measured three times daily. Three replicate plates were set up for each treatment, and the experiment was repeated twice. The implication of the results was expressed as PIRG that considers the percentage inhibition of radial growth expressed as: %PIRG=((R1-R2))/( R1)*100, where: PIRG = percentage inhibition of radial growth; R1 = average value of the radial growth of the target in the absence of the antagonist (control); R2 = average value of the radial growth of the target in the presence of the antagonist. The experiments were repeated using other media (malt extract agar, minimal medium agar and Czapek-agar) but the best results were observed in PDA medium.
To investigate the bioactivity of Trichoderma against target microorganisms different tests were performed:
i) volatile test results of T. harzianum T22 and T. asperellum B1 against Fusarium oxysporum; ii) volatile test results of T. harzianum T22 and T. asperellum B1 against Rhizoctonia solani; iii) volatile test results of T. harzianum T22 and T. asperellum B1 against Rhizoctonia solani.
The presence of Trichoderma harzianum T22 and Trichoderma asperellum B1 induced an inhibiting effect on the target Fusarium oxysporum that probably was due to the production of volatile compounds since the hyphal contact was avoided in this test. Percentage inhibition radial growth (PIRG) of F. oxysporum in the presence of Trichoderma harzianum T22 was 49.4% while in the presence of Trichoderma asperellum B1 was 32.8%. The presence of Trichoderma harzianum T22 and Trichoderma asperellum B1, instead, induced a weak inhibiting effect on the target Rhizoctonia solani that probably was always due to the production of volatile compounds. Percentage inhibition radial growth (PIRG) of R. solani in the presence of Trichoderma harzianum T22 was 30.7% while in the presence of Trichoderma asperellum B1 was 12.5%. The presence of Trichoderma harzianum T22 induced an inhibiting effect on the target Pythium ultimum that we quantified with the PIRG value 30.9%. In contrast, Trichoderma asperellum B1 induced a very low inhibiting effect on Pythium and in this case the PIRG value was 4.7%. GC-MS in combination with solid-phase microextraction (SPME) will be used to explore the volatile secondary metabolites released by two fungal species grown on PDA. Extraction of the volatile substances will be performed by using a SPME system into the head space of the Erlenmeyer flask. Inside a cannula, the fibre will be passed through a septum and exposed to the volatiles derived from the cultures. After adsorption for 24-48 h, the fibre will be retracted into the needle and removed from the flask to perform gas-chromatographic analysis.
In addition, other studies were carried out on:
biological control of the Rhizoctonia solani and Pythium ultimum disease complex on GF rootstocks, using mixtures of Trichoderma harzianum T-22 with Trichoderma asperellum B1, Trichoderma viride and Pseudomonas fluorescens F113, during the acclimatisation period.
Disease severity: Trichoderma harzianum T22 combined with Trichoderma asperellum B1 significantly reduced the Rhizoctonia solani and Pythium ultimum mediated disease severity. In contrast, with the Trichoderma harzianum T-22 mixtures with Trichoderma viride and Pseudomonas fluorescens F113, there were no significant differences when compared with the positive control plants. The treatments seemed to be ineffective against the specific disease complex.
Plant growth promotion: compared to the control plants, Trichoderma harzianum T22 combined with Trichoderma asperellum B1 seemed to promote the plant growth characteristics measured (shoot weight, root weight and number of leaves). Among the treatments of the specific experiment, Trichoderma harzianum T22 combined with Pseudomonas fluorescens F113 showed increased levels of all plant growth characteristics only in the absence of the plant pathogens.
Colonisation experiments: Trichoderma harzianum T22 combined with Trichoderma asperellum B1 seems to be an excellent coloniser of the GF root system. There was no sign of competition between those two biocontrol agents, moreover the outcome from the interaction between them appeared as enhancing colonisation. Root samples taken two days after transplantation were 80% colonised by the specific biocontrol agents. The pronounced treatment revealed increased colonisation rates during the first ten days after transplantation - the crucial period for plant protection and disease severity - compared to Trichoderma harzianum T22 combined with Pseudomonas fluorescens F113 (moderate colonisation rates) and Trichoderma harzianum T22 combined with Trichoderma viride (low colonisation rates). In the turf colonisation experiments, the biocontrol treatments showed similar results. Specifically, the mixture of Trichoderma harzianum T22 and Trichoderma asperellum B1 was present in all three dilutions of the turf samples and was statistically differentiated compared to Trichoderma harzianum T22 combined with Pseudomonas fluorescens F113 (moderate turf colonisation activity) and Trichoderma harzianum T22 combined with Trichoderma viride (low turf colonisation activity).
WP3 - Optimisation of biotisation techniques on micropropagated rootstocks.
The aim of this WP3 was to define and optimise the most appropriate inoculation time and method, and to verify the effects of mycorrhysation on micropropagated rootstocks.
T.3.1 Optimisation of techniques to inoculate micropropagated rootstocks in the different growth stages.
We estimated: i) the effects of five different peat types: Kekkila, TS1, Klasman Plug Mix (Mix), Klasman Gold (Gold), and Havita; ii) the effects of treating or not with Trichoderma harzianum T22 and Glomus intraradices and Agrobacterium radiobacter K84.
The different method of inoculum applications were: a) mixing T. harzianum or G. intraradices with peat and incubating for a week before planting; b)mixing T. harzianum into peat and incubating for 20 days before planting; c) dissolving the inoculum into a solution and dipping the plants just before planting (dipping method). This was applied for Trichoderma and Glomus and A.radiobacter.
The rootstocks used were GF677, OHF19-89, Myrobolan 29C and GiSeLa® 6.
Concerning the different methods of inoculation applied it was concluded that the dipping method is better than the mixing one. This result has a positive impact on optimisation of rootstock biotisation techniques and also on reduction of the production costs for the SME. The dipping method is simpler, more economical and more efficient and increases the inoculum presence. Four weeks after inoculation, the G. intraradices root colonisation was not observed. Results showed that inoculation occurs later. In Italy, Glomus was detected after 6 months from inoculation. In general, regarding the effects of different peats on rootstocks inoculated with Glomus, Kekkila showed the best results for numbers of leaves, heights, shoot dry weights, except for GF677 rootstocks in which Havita peat performed better. Instead, Gold peat gave the best results for all rootstocks on root dry weight and TS1 on root and shoot ratio.
T.3.2 Analysis of symbiotic effects on plant growth and quality in the nursery.
The main objective was to study the effect of the mycorrhization on growth and plant quality as well as on the efficiency of nutrient uptake during rootstock development in the nursery.
During the reporting period, all those effects were evaluated in two different management systems. Conventional nursery management (Nursery MNGT) were compared with a sustainable one (Sitinplant MNGT). For this experiment were used micropropagated and inoculated (Glomus, Trichoderma and A. radiobacter) and not inoculated rootstocks produced in Greece during the acclimatisation phase and transferred to the nursery. Two additional treatments with Streptomyces lydicus WYEC 108 and Bacillus subtilis QST 713 were also applied. All trials were carried out in Bulgarian and Turkish experimental fields.
Results, obtained during last sampling in Bulgaria, showed that: i) rootstocks of GiseLa® 6 treated with Glomus gave the highest values of stem diameter and total dry matter compared to all other treatments. In addition, high stem diameter values were found in Trichoderma + Bacillus treatments; ii) Control Sitinplant treatment in OHF19-89 and Myrobolan 29C rootstocks showed the best values of stem diameter and accumulation of total dry matter. Moreover, in all the other biometric parameters measured the highest values were found with Trichoderma+Bacillus subtilis as well as Trichoderma+Streptomyces lydicus treatments; iii) for GF677 highest total dry matter resulted with Control Nursery treatment followed by the Glomus + A. radiobacter one. The analyses of plant mineral uptake were done in May, July and October 2009. Three plants (replications) were destroyed and analysed per treatment. Each plant was separated into root, stem and leaf parts. Each analysis was performed with 180 samples for mineral uptake analysing basic macro and microelements (N, P, K, Ca, Mg, Zn, Cu, Mn, Fe). The results obtained showed that the best uptake of macro elements was achieved by the rootstocks treated with microorganisms (Sitinplant management) compared to the plant control managed with conventional nursery techniques. Maximising the nutrient uptakes (N and K) by microorganism applications, we can reduce the risk of leaching losses of these elements into the ground water and so reduce the environmental impact. Groundwater pollution caused by agricultural activities is a serious problem in many regions of the world.
Analysis of persistence and presence of mycorrhizae. For each rootstock and treatment, five root samples were collected and used to estimate mycorrhizal colonisation (Glomus and Trichoderma). Arbuscular mycorrhizal colonisation analysis showed higher percentage of Glomus root colonisation in GiSeLa® 6 treated with Glomus. For GF677, Agrobacterium radiobacter was found in the plants that were inoculated with A. radiobacter combined with G. intraradices. Bacillus subtilis and Streptomyces lydicus, from the additional field treatments, were presented in sufficient quantity as it was expected. In particular, Bacillus subtilis was detected in GiSeLa® 6 and OHF19-89 rootstocks.
In Turkey, analysis of fresh and dry matter accumulation were carried out on ten plants (July 2009) for each rootstock and treatment, with a total about 190 samples examined. Determinations of macro and microelements were carried out on each rootstock treated in two separate samplings September and November (2009) with a total of 180 plants examined. In addition, specific mineral content analyses were carried out on each plant sample separating leaves, shoots and roots.
Inoculated roots were observed in all experimental plants denoting the success of the procedure.
Twenty plants per treatment and per rootstocks were selected randomly in 6 different sampling periods. On all collected plant samples, different biometric parameters were measured: plant height, number of leaves, stem diameter, number of lateral shoots and length of lateral shoots.
WP4. Optimisation of micrografting techniques on biotised rootstocks.
FITO SME produced micropropagated and biotised rootstocks to be used in grafting or micrografting tests and further experimentation. Plant material was transferred to the nurseries (Italy, Turkey and Bulgaria). All SMEs set up the grafting or micrografting method.
T.4.1 Optimum period for micrografting techniques.
To optimise and therefore improve the potential of the micro-grafting techniques, the SITINPLANT project will focus on the following experimental scientific issues:
i) optimisation of the micro grafting process on biotisated rootstocks with the aim of increasing the percentage of grafting success and plant quality;
ii) increase our understanding of the physiological and morpho-anatomical relationships of the grafting process in relation to biotisation;
iii) development of innovative techniques to reduce failures or delays in bud break in relation to biotisation.
The research compared the following possibilities:
i) micrografting in October-November, using actively-growing micropropagated rootstocks and buds of the cultivars to be grafted from mother plants in the same period;
ii) micrografting using buds of the cultivars to be grafted taken from mother plants in December-January and kept in a refrigerator and micrografted in February-April on actively-growing micropropagated rootstocks.
The research was carried out on the most commercially interesting fruit tree species (Apricot, Peach, Cherry, Pear), for each species using combinations of rootstock and scion to allow an accurate evaluation of the entire process. Plant material, micropropagated and treated with biological agents (see previous deliverables, about 2000 rootstocks) was transplanted to 0.9 L square pots with substrate mix TS1 (Klasmann-Deilmann Gmbh-Lithuania) at AGTE SME - Italy (July 2009). Buds were micrografted onto rootstocks in October 2009 and April 2010. Micrografting combinations: scions of Portici, Sunburst and Williams were supplied by the nursery "Iocoli" at Santarcangelo, Potenza-IT; Big Bang and TC Sun by the nursery "Vitroplant" at Cesena-IT. Preliminary trials indicate that "chip budding" is the best technique for obtaining higher grafting percentages also for reducing the scion material needed. To determine the most suitable time to perform micro-grafting, the rootstock Gf677 was grafted by chip budding in October using buds from scions of the same period. Also, in spring using buds from scions selected during winter and stored at 4°C.
Comparing the data obtained from the two grafting periods, the best performance was clearly shown to be in October. Autumn (October) is the most suitable period for micro-grafting, using mycorrhisated rootstocks, actively growing in pots under controlled conditions and fresh buds selected during the same week. The grafting efficiency is closely linked to cambial differentiation and callus formation. Unsuccessful micrograftings were shown to be associated with three main causes:
i) by the operator's technical skill in choosing the right material (diameter, quality, etc.);
ii) by the healing rate of the cut rootstock tissues and by the different growth rate between cultivar and rootstock meristems: when the rootstock is too vigorous, cultivar buds can be pushed out and die;
iii) by sanitary problems occurring when scions are stored in a refrigerator (mould formation).
T.4.2 Effect of biotisation of rootstocks on micrografting success.
This Task 4.2 aims to increase the grafting percentages in the micro-grafting technique using biotisated rootstocks. The related experiment evaluates the positive effects of biotisation on the micrografting success rate.
Grafting percentages. Comparing the data obtained from each rootstock/cultivar combination (micrografts done in October 2009), the micrografting technique and, in particular, chip budding were revealed to give good results in peach, 88% of total grafts took. Considering all combinations tested, the efficiency of the technique seems to be related to species sensitivity. Only GF 677 and peach showed interesting results. Unsuccessful micrograftings could be due to the healing speed of the cut rootstock tissues and by the different growth rates between cultivar and rootstock meristems.
Mycorrhization effects. Overall, rootstock mycorrhisation did not seem to affect the grafting potential of each species combination directly. There were no significant differences among treatments and controls on grafting success. The efficiency of micrografting is closely linked to cambial differentiation and callus formation.
T.4.3 Effect of biotisation on grafting success of the plants in the nursery.
Mycorrhizal fungi have been shown to increase uptake of water and mineral nutrients by plants. Until now, there have been no clear relationships between mycorrhizal inoculation and grafting/budding success. The objective of this research was to record the effect of biotisation on grafting success of the plants in the nursery.
All experiments were carried out in Turkish and Bulgarian field sites to study the following topics:
i) effects of biotisation on grafting success rates;
ii) influences of biotisation on physiology of grafted plants.
All analyses were carried out under one or other of two different field management systems:
i) conventional nursery management;
ii) sustainable Sitinplant management.
GiSeLa®6 rootstock grafted with cherry cultivar Van: the best rates were generally found with the Trichoderma biotisated plants which exceeded the controls by between 3% and 11%. OHF19-89 rootstock grafted with the pear cultivar William: compared with the results obtained for the other experimental rootstocks, the best rates of up to 60%, were achieved with the Glomus + Trichoderma combination. As with the previous rootstock, there were no significant differences for the additional bacterial treatments. Myrobolan 29C rootstock grafted with the plum cultivar Angeleno: the best rate was for the Glomus + Trichoderma variant. GF 677 rootstock grafted with the peach variety Royal Glory: there were no significant differences between mycorrhizated and non-mycorrhizated variants in the SITINPLANT plots.
Project experimental field - Bursa, Turkey. The micropropagated material obtained from Fitotechniki (Greek-SME partner of the SITINPLANT) was planted on 26 June following standard procedures, after agro technical soil cultivation and setting up for drip irrigation. Project treatments were compared to classical nursery applications. Two antagonistic microorganisms (Bacillus subtilis and Streptomyces lydicus) were used. The number of plants used in the trial at Bursa was about 13500.
Vegetative buds were T-grafted onto rootstocks treated with biological agents in September-October 2009.
The percentages of successfully grafted plants (shooting) were observed and calculated for each variety in a 4x3 table (levels of mycorrhizae and bacteria). Chi-square tests were performed on observed and expected numbers of grafting results in three different ways:
1) bacterial treatments were combined for each mycorrhizae treatment; and mychoryzae treatments versus grafting results (success/failure);
2) mycorrhizae treatments were combined for each bacteria treatment; and bacteria treatments versus grafting results (success/failure);
3) bacteria-mychoryzae combination treatments versus grafting results (success/failure).
WP5. Optimisation of growing techniques.
The general objective of this WP5 was to define the correct nursery techniques management that are compatible with microorganism symbiosis and that ensure the most favourable conditions to obtain good quality of micrografted plants in pots and grafted plants in the nursery.
At this stage plant material was prepared with about 975 rootstocks micro-grafted in Italian nurseries, 7879 in Bulgaria and 7894 in Turkey.
The WP5 aimed at studying:
a) persistence of microorganisms on micrografted plants in pots (T.5.1);
b) effects of different managements in the nursery on persistence of microorganisms of grafted plants on inoculated and non inoculated rootstocks (T.5.2);
c) evaluation of the growth and quality of micrografted plants grown in pots and grafted plants in the nursery (T.5.3).
T.5.1 Persistence of microorganisms on micrografted plants grown in pots.
UBAS conducted trials under controlled pot conditions according to the existing AGTE SME production processes. The first step was to check for microorganism persistence. Plant material, micropropagated and treated with biological agents was transplanted into 0.9 L square pots with substrate mix TS1 (Klasmann-Deilmann Gmbh-Lithuania) at AGTE SME - Italy (July 2009). Buds were micrografted onto rootstocks in October 2009 and April 2010. Plants were grown under 50% shade net. Water was automatically distributed by overhead mini-sprinklers (range 2.5 m, 120 L/h) with three applications during July and August and two in September and October 2009. Fungicides were not used to avoid detrimental effects on the biocontrol agents. Rootstocks were treated with Actara (Thiamethoxam), 40 g/hL; grafted plants with Confidor (Imidacloprid), 50 ml/hL to control aphid infestations. A basic Scotts (20-20-20 + micro), 0.5 kg/hL and potassium sulfate 1 kg/hL were applied twice by fertigation in September 2009 before grafting. Different foliar fertilisers 31-11-11, 330 g/hL plus 330 ml/hL of organic N 5%, and 18-6-2+12, 0.186 g/pot were applied respectively in April and three times in May 2010. Chip budding was done as described.
Presence and persistence of microorganisms. The persistence of bio-control agents was checked on 10 plants per treatment, at the end of the production cycle in June 2010. Qualitative and quantitative measurements were carried out on roots: infection and arbuscular percentages, intensity of colonisation for Glomus; and on substrates for CFU Trichoderma detection.
There were no significant effects of mycorrhization on graft take.
Glomus intraradices. Considering the frequency of system mycorrhisation (F%), treated plants showed the best results, 100%, in all four rootstocks, compared to controls. Controls were probably infected by natural agents always present in the soil or in the irrigation water. For all treated plants, colonisation intensity (M%), the most important parameter to evaluate the colonisation, ranged between 40 and 85%, reaching the highest values in micrografted Gf677, compared to controls; the differences were statistically significant. The arbuscular presence (a% and A%) was evident in Glomus treated Gf677, about 50%, in Myrobolan 29C plants, 10%, and in OHF19-89, about 20%. Arbuscular differences were, however, less marked, above all in GiSeLa®6.
T.5.2 Effect of different managements in the nursery on persistence of microorganisms of grafted plants on inoculated and non inoculated rootstocks.
AUTH partner monitored the persistence in the field under nursery management, or modified Sitinplant management of microorganisms used to inoculate plants at the rooting phase and again at transplanting in the Bulgarian and Turkish experimental fields. FITO SME produced micropropagated and biotised rootstocks to be used in grafting tests and further experimentation. Plant material was transferred to the nurseries (Turkey and Bulgaria). All SMEs set up the grafting and management methods. Soil and root samples were collected from the rhizosphere of the field plants. Analysis for arbuscular mycorrhizal fungal root colonisation from the Turkish samples was conducted in Turkey.
Trichoderma harzianum. For T. harzianum two different media were used. Malt extract agar is non-specific to Trichoderma sp, and we have previously used it for detection of T. harzianum, however its lack of selectivity made quantification difficult: other fungi would also grow on this medium and as a result only qualitative data were possible (presence/absence), dividing the number of positive plates with the total number of plates/replicates. For quantitative data a semi-selective medium was employed, however, until sporulation (required for positive identification) the fungus may also spread on the plate and colonies may overlap, therefore on one occasion qualitative rather than quantitative data were reported with this medium also. We must note that almost no Trichoderma was recorded in both control treatments and media used, particularly in the nursery management plots. Initially, there was nothing detected in Sliven (Bulgaria) in October 2009 with the semi-selective medium, and numbers remained low in the Bursa (Turkey) samples of December 2009. However, the Trichoderma population increased in 2010 and peaked in October 2010 Sliven rootstock rhizosphere. Although there was high variability in the results, and no discernable pattern regarding interaction of T. harzianum with the other organisms applied, and taking into account results from both media, the form of the inoculum does not seem to make a difference to T. harzianum persistence. However, there was some indication from Myrobolan 29C and the semi-selective medium that Trianum P gave slightly higher numbers than Trianum-G after re-application. In addition, due to data variability, and appearance and disappearance of the organism with time with no perceptible pattern, re-application, regardless of inoculum form, does not seem to offer any particular benefit to persistence of T. harzianum.
The molecular identification data confirmed that most of the isolates were Trichoderma harzianum T22. In Bulgaria all (100%) of the isolates were T. harzianum T22, and in Turkey (62%) of the isolates, with local Trichoderma sp. also being present. On the basis of their morphological characteristics or due to molecular analysis of the internal transcribed spacer 1 and 2 regions (ITS1 and ITS2), Trichoderma virens (25% of the isolates obtained) and Trichoderma spirale (13% of the isolates obtained) were also present.
T.5.3 Evaluation of the growth and quality of micrografted and grafted plants.
Evaluation of the growth and quality of micrografted plants grown in pots.
UBAS conducted trials under controlled pot conditions according to the existing AGTE SME production process. In particular, UBAS studied:
a) effects of symbiosis on micrografted plant growth and quality:
analysis of dry weight of micrografted and grafted rootstock roots system; plant growth; analysis of dry matter accumulation and partitioning between different plant organs; analysis of total mineral elements uptake of micrografted rootstocks in pots;
b) effects of symbiosis on plant water uptake and root conductivity.
Effects of symbiosis on micrografted plant growth and quality. Dry weight of micrografted plants at the end of the production cycle (plants ready for the market). Glomus and Trichoderma biotised plants reached total mean dry weights of 1.46 and 2.21 g greater than the controls (higher by 19.6 and 29.8% respectively). Differences between the two treatments were not significant; but it is necessary to stress that, although there was a high variability for each treatment, plants inoculated with Trichoderma grew on average about 1 g more than the Glomus ones (8.5% more). The plants of Big bang/GF677 and TC Sun/Myr. 29C combinations reached a dry weight of 11.96 and 10.58 g, and had diameters of 4.57 and 4.66 mm, respectively; while the Sumbuster/GiSeLa®6 and Williams/OHF 19-89 combinations grew less, showing mean values of total dry weight of 7.46 and 4.58 g respectively. The growth ratio between the scion and rootstock of the different combinations emphasised the significant differences. In particular, while for the combination Big bang/GF677 the dry weight ratio was 0.97 i.e. there was a perfect linear growth rate of bionts, for the other three combinations was clear the predominance of the rootstock, with a greater growth of 1.23 1.76 and 2.30 g, respectively in Williams/OHF19-89, TC Sun/Myr. 29C and Sunbuster/GiSeLa®6.
The analyses of dry weight data for each combination confirms less control growth compared to inoculated plants; except for the combination William/OHF19-89, in which difference were not significant. There was a mean growth of about 10% greater in Glomus plants for the Big bang/GF677 combination, while, for the other combinations, Trichoderma treatment induced the best results (plants were about 20% bigger than the controls).
Evaluation of growth and quality of grafted plants in nursery.
The most important step for practical implementation of micropropagated, inoculated and grafted rootstocks in the nursery was a complex study of the interaction between mycorrhizae and antagonist biocontrol micro-organisms on plant growth and quality, as well as on the efficiency of nutrient uptake and disease incidence during the whole vegetation period. At the same way, the types of grafting as well as grafting compatibility, between rootstock and scion, were considered. Environmental conditions during the experimental period had a big influence on the above mentioned complex of interactions.
In vitro micropropagated and biotisated rootstocks were obtained from SME Fitotechniki - Greece. They were planted in the Bulgarian project experimental field (Sliven region) on 16 April 2009. The rootstocks were grafted with the cultivar scions on 14-15 and on 16 August 2009 by T-budding, 10 cm from the soil. In Turkey the planting was performed on 26 June 2009 by standard procedure, after the agrotechnical soil cultivation as well as the construction for drip irrigation.
The following experiments were carried out with successfully grafted plants of rootstock/scion combinations. Distribution of experimental plants was done on randomised plots, related to the project experimental plan for 2010, including additional mycorrhizal and bacterial treatments with the same bio-agents in some of the GF677/Royal Glory combinations. Additional treatments were done inside the experimental plots with the same mycorrhizal inoculi used in the first stages of in vitro rootstocks production in Fitotechniki, Greece. Regarding the positive results obtained in 2009 with Trichoderma/Baccillus subtilis and Trichoderma/Streptomyces lydicus treatments, both Trichoderma inoculations were also included as different combinations. All microorganisms were applied manually, as close to the roots as possible. On the basis of previous results, the amount of nitrogen and potassium was increased by 30%. The fertilisers in the SITINPLANT plots were applied proportionally by fertigation (approx. 2 times per month, depending on rainfall), altogether 12 times during the whole vegetative period. The distributions of fertilisers in the conventional SME plots were done proportionally twice during the vegetative period by soil implementation and leaf feeding. Growing parameters were measured: height (rootstock upper part + scion) and stem diameter (scion, 10 cm from the grafting point). The analyses of dry matter accumulation were done at the start and at the end of the plant vegetation cycle, during the months June and October respectively. Three plants were destroyed and analysed per treatment. Each plant was separated into four parts: rootstock root, rootstock upper part, variety shoot, variety leaves. Fresh/dry matter accumulation analyses were done for each plant part. The analyses of mineral uptake were done twice in the vegetation period (June and October 2010). Each sample was checked for mineral uptake of the 6 basic macro and microelements (N, P, K, Ca, Mg and Fe).
Phytonematodes. After two years of experiments, mycorrhizae contrasted with pathogen development; treatments showed better results than the controls with a reduction in the number of ectoparasite phytonematodes under nursery conditions. Moreover, the density of semi-parasite and free living ones was maintained. It means mycorrhizal treatments can be strongly recommended as a tool for biological control.
Biometric analyses. Results obtained after the second vegetative period can be summarised as:
i) Glomus treatment appeared to induce the best growth for GiSeLa®6/Van experimental plants;
ii) Trichoderma experimental plants showed best height results in OHF19-89/William rootstock/scion combination. All variants demonstrated aligned and good plant growth with regard to stem diameter but without significant differences;
iii) Glomus+Trichoderma variant induced the best height growth rate in both the controls with 12% (Control SITINPLANT) and 17% (Control Nursery) in the Myrobolan29C/Angeleno combination. No remarkable differences were measured in stem diameter among all the experimental plots;
iv) additional treated variants Glomus (AG) and Glomus+Agrobacterium (AG) provided best final results in GF677/Royal Glory experimental plots.
Fresh/dry matter accumulation. The GiSeLa®6/Van: Glomus variant clearly had the best fresh and dry matter accumulations as well as FW/DW ratios. OHF19-89/William: in this case, the Trichoderma variant showed the highest fresh and dry matter accumulation. Myrobolan29C/Angeleno: Glomus+Trichoderma and Glomus variants provided the highest fresh and dry matter values and fresh/dry matter ratios. GF677/Royal Glory: Glomus treatments influenced the fresh and dry matter accumulation as well as FW/DW ratios. The positive effects of additional field mycorrhisation were confirmed.
Basic mineral elements uptake. GiSeLa®6/Van: both Glomus and Trichoderma variants showed the best mineral uptake rates, where Glomus clearly exceeded in N and Fe accumulations and Trichoderma in Mg. Concerning K and Ca mineral elements Glomus and Trichoderma experimental plants showed very close accumulation rates. OHF19-89/William: in all cases the Trichoderma variant was demonstrated to have very good accumulation activity. Myrobolan29C/Angeleno: Glomus+Trichoderma for the rootstock part and Glomus for the scion part presented the best final absolute values. GF677/Royal Glory: Glomus+Agrobacterium induced the best results. Additional treatments gave an advantage in all cases.
WP6. Definition and evaluation of innovated process.
The main outcomes are working protocols and specific technical procedures containing information related to the phases of the optimised cycle and outlining possible critical points.
T.6.1 Production scheme of micropropagated and biotisated rootstocks.
In advanced agricultural systems, grower performance is closely tied up with nursery propagation efficiency. To respond to modern plant production requirements, nurseries must combine economic and environmental aspects. New process knowledge is important to define the innovated plant production describing the criteria for optimising micropropagation and biotisation methods in rootstock production and streamlining nursery organisation.
The company Fitotechniki (FITO), in collaboration with AUTH, had the responsibility in the Project of developing the protocol for production of the inoculated and micropropagated rootstocks, estimating the qualitative, environmental and economic benefits.
The inoculation by dipping, on the basis of experiments carried out, was more efficient. Whereas the possibility to propagate fresh culture of T. harzianum, results will be better. Trichoderma is an excellent root and peat coloniser resulting in promotion of specific plant growth characteristic and suppression of P. ultimum and R. solani mediated disease.
T.6.2 Production scheme of micrografted and grafted fruit tree plants using inoculated micropropagated rootstocks grown in pots and in the nursery.
The protocol for fruit tree production was developed by the Italian, Bulgarian and Turkish partners of the Project, in collaboration SME-RTD, estimating the qualitative, environmental and economic benefits gained.
Micrografted fruit trees. A new technical protocol was defined at the end of the Italian trials as a result of the two-year experimental activity. The production scheme is developed for the rootstocks GiSeLa®6, OHF19-89, Myrobolan29C and GF677. Scions Portici, Sunburst and Williams were supplied by the nursery "Iocoli" at Santarcangelo, Potenza-IT; Big Bang® and TC Sun by the nursery "Vitroplant" at Cesena-IT.
Rootstocks are obtained by micropropagation and delivered to the nursery at the end of the summer (height about 15-20 cm, diameter 2-3 mm). Plants in the nursery are transplanted in square pots (size: 9x9 cm, 13 cm height) that avoid root circling and maintain best growth to the end of the process, adding white peat and a complex slow-release fertiliser. It is important that the mix is poor in phosphorus and that this element is not applied during regular fertilisations or it could interfere with the symbiotic fungi. In the same way, systemic fungicide use should be avoided. For the micrografting, the "chip-budding" technique is used; it should be carried out in the period September-October. Production efficiency defined grafts 50-60 plants/hour/person.
The few days following the grafting are extremely tricky, in particular it is important to assure temperatures of 25-28°C and RH of 80-90%. Plants are maintained in a protected environment and under optimal water/nutritional/sanitary conditions. In April plants are ready for the market, potted plants with a height of about 40-50 cm and a diameter of 4.5-5 mm. The cycle previously proposed was best in terms of plant quality and efficiency in grafting take. But it is possible to develop different processes. These alternative cycles can apply to specific commercial needs. It is possible to use stored buds (sampled in the previous year), reducing the production cycle, from 9 months of the first proposed to 4-5 months. The production cycle under greenhouse conditions is characterised by high flexibility and may be started at any time of year.
Grafted fruit tree plants. The production scheme presented is based on the results from two-year experiments carried out in the Bulgarian and Turkish project experimental fields. It could be implemented in other environmentally suitable regions in Bulgaria and Turkey with some adjustment, mainly in the fertilisation plan, depending on preliminary soil and water analyses. The production scheme is developed for the rootstocks GiSeLa®6, OHF19-89, Myrobolan29C and GF677. The scion varieties grafted on them in Bulgaria were respectively cv. Van (cherry), cv. William (pear), cv. Angeleno (plum) and Royal Glory (peach), in Turkey cv. Skeena, cv. Black Diamond and cv.s Angeleno, Royal Lee and Tardired, for the SME applications, GF677 was grafted with the cultivars Tardired and Royal Lee. The rootstock should be virus-free, in vitro obtained and treated twice with Trichoderma harzianum (T22 strain) and Glomus intraradices (separate and in combinations) at the beginning of the acclimatisation stage and before transplanting in the nursery.
Potential Impact:
The project success will allow production of micrografted and grafted plants using inoculated micropropagated rootstocks. The following main benefits are achieved:
-Improvement of plant tolerance to biotic and abiotic stress through the establishment of a long-lasting plant symbiont colonisation of root and leaf surfaces;
-Reduction of chemical pesticides used in the nursery and orchard through the induction of a systemic resistance;
-Reduction of the use of chemical fertilisers by stimulating root development and increasing uptake efficiency through natural agents;
-Reduction of agricultural environmental impact by treating unhealthy soils through inoculation of plant symbionts which decrease the activity of deleterious soil-born microflora and inactivate toxic compounds in the root zone;
-Reduction of costs and time of the nursery production cycle.
Production scheme of micropropagated and biotisated rootstocks.
As a result of the new production scheme plants are healthier, and there are very few losses, due to more efficient suppression of soil pathogens, in the acclimatisation phase. In fact, previously, using fungicides, the company FITO was losing about 10% of the plants during the acclimatisation phase while now, with the application of antagonists, only a small percentage (approximately 3%) is lost. In addition, plants are more homogeneous in development, and there is a shortening of the hardening period by one week, which translates to worthwhile savings in personnel and greenhouse space. With the previous production scheme pesticides were used pre-emotively, but are now not included in the current scheme: this is an enormous benefit for the personnel, the SME and the environment. Previously the personnel had to use the pesticides, and re-entrance to the greenhouses was forbidden for some time after application. Now exposure to chemicals and the associated health effect risks are minimised and staff feel safer in a healthier environment. Environmentally, the risk of contaminating the soil and the groundwater with chemicals is minimised. There is also a financial benefit. The cost of pesticides with the previous scheme was 5500 Euros per million micropropagated plants produced (current production of the SME is about ten million plants per year), while the cost of applying and using the microorganisms for the same number of plants is 1400 Euros (using commercial inoculum).
Production scheme of micrografted fruit trees in pots.
To estimate the impact of the project results application, not only should the quantitative cost reduction of the overall production be taken into account but also the improvements in plant quality using environmentally friendly techniques that SITINPLANT has achieved. The overall production cost reduction is estimated at for 35% for bi-member plants of 30-40 cm height and of about the 25% for producing micrografted plants of 80-120 cm height. The project results, with further RTD activities, can also be transferred to other sectors such as the forest sector, in particular for the recovery of areas destroyed by fire or natural calamities and for sectors such as specialised greenhouse floriculture. Moreover, all sectors related to agriculture, such as food industry, will be positively affected by the project results indirectly. Another relevant aspect of this project is the environmental impact, since the inoculated and micrografted plants reduce the input of chemical fertilisers and pesticides, reducing soil and water pollution.
Production scheme of grafted fruit tree plants.
Traditional grafting processes are characterised by variability, low scion affinity, poor plant development, low quality, susceptibility to pathogens, non-certified plants etc. SITINPLANT management provides a competitive price, the overall production cost reduction is estimated at 30% of the traditional cost, in addition to the listed advantages in terms of innovation and quality plant production. Other main outcomes and benefits for SME in Bulgaria and Turkey are that SITINPLANT management allows a drastic reduction of the application of N and K by 71% and 86% respectively and 100% for P in the first year. In the second year nursery cycle, reduction percentages are respectively 41%, 56% and again 100% for P. Soil-born pathogen control was found to be possible through inoculation with antagonist microorganisms Baccilus subtilis and Streptomyces lydicus which is an enormous advantage from an economic and environment point of view. The priority use of biological technologies will facilitate and reduce the time for the transition from conventional to organic farming for SMEs. In addition, implementation of the SITINPLANT management production scheme could improve the social status of the people living in Bulgaria and Turkey affected regions.
A detailed IPR and knowledge management plan was worked out where the IPR on the different project results was defined. The IPR and knowledge management addresses the following issues:
-ownership of the project results (foreground knowledge);
-licensing of pre-existing know-how;
-transfer of the knowledge gained within the project;
-confidentiality of project results and dissemination strategy.
This is due to the following consideration:
-It is very difficult to differentiate the use of such knowledge among the different companies;
-SMEs involved are not competitors (they operate in very different markets);
-The level of investment for each SME is considered fair for the market in which it operates and for its specific expected return.
Specific agreement on Intellectual Property was undertaken with the RTD performers. This was based mainly on the following rules:
-RTD providers are able to realise publications and present the results obtained during the project in relevant international conferences but the information to be provided should not be sensitive information.
-The results of the project are property of the SMEs involved. The handling of the Intellectual Property Rights was defined through the Consortium Agreement that proposers created and signed before the end of the first month of project activity.
The objectives of the Dissemination phase are:
i) to disseminate information about the project, its objectives, its approaches and its results;
ii) to facilitate collaboration and information exchange between relevant research and agricultural communities;
iii) where applicable to promote the use of techniques resulting from the project amongst the target groups: research entities, SITINPLANT stakeholders, others;
iv) to create two-way communication channels with stakeholders, research communities and industry for disseminating the project deliverables and conclusions;
v) to ensure that the products of the project live on in a commercial context, this way assisting the Exploitation of the project results.
Conclusions
Impact for the SME participants
The project success has clearly improved SMEs competitiveness as far as project results which were targeted mainly to the nursery plant sector - the core business of all SMEs proposers.
Several advantages, in terms of increased competitiveness are foreseen for SMEs proposers:
-Reduction of production cycle times: from 2 years to 3-8 months.
-Increased tolerance of the plants produced: with the new techniques plants will be more tolerant to pathogens and will make more efficient use of the mineral and water resources of the soil.
-Reduced production costs: through the use of biotised and micrografted plants.
-Increased know how provides several direct and indirect commercial benefits, also taking into consideration consulting services that can be offered to implement the innovative techniques.
Improvement of industrial competitiveness across the European Union
The plant nursery sector plays a very important role in the wider agriculture sector, since quantity and quality of yield depend mostly on the quality of the plant material used for orchard plantation. This sector, as well as the wider agriculture sector in Europe is currently experiencing competitiveness problems due to the emergence of global competitors able to offer aggressive commercial policies. For this reason it is essential for European SMEs to innovate their production processes to remain competitive and to comply with environmental policies.
Employment opportunities
The increased competitiveness of SMEs in a growing market such as fruit tree nursery sector (about 30%) produces new employment opportunities (20-30 new workers, above all women, during the project).
European Cohesion
The project proposes a strong collaboration among partners coming from four European countries, thus improving European cohesion.
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